Tumor elimination by clustered microRNAs miR-306 and miR-79 via noncanonical activation of JNK signaling

  1. Zhaowei Wang
  2. Xiaoling Xia
  3. Jiaqi Li
  4. Tatsushi Igaki  Is a corresponding author
  1. State Key Laboratory of Biocontrol, School of Ecology, Sun Yat-sen University, China
  2. Laboratory of Genetics, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Japan
  3. Guangzhou Key Laboratory of Insect Development Regulation and Application Research, Institute of Insect Science and Technology & School of Life Sciences, South China Normal University, China

Abstract

JNK signaling plays a critical role in both tumor promotion and tumor suppression. Here, we identified clustered microRNAs (miRNAs) miR-306 and miR-79 as novel tumor-suppressor miRNAs that specifically eliminate JNK-activated tumors in Drosophila. While showing only a slight effect on normal tissue growth, miR-306 and miR-79 strongly suppressed growth of multiple tumor models, including malignant tumors caused by Ras activation and cell polarity defects. Mechanistically, these miRNAs commonly target the mRNA of an E3 ubiquitin ligase ring finger protein 146 (RNF146). We found that RNF146 promotes degradation of tankyrase (Tnks), an ADP-ribose polymerase that promotes JNK activation in a noncanonical manner. Thus, downregulation of RNF146 by miR-306 and miR-79 leads to hyper-enhancement of JNK activation. Our data show that, while JNK activity is essential for tumor growth, elevation of miR-306 or miR-79 overactivate JNK signaling to the lethal level via noncanonical JNK pathway and thus eliminate tumors, providing a new miRNA-based strategy against cancer.

Editor's evaluation

This article is valuable as it uncovers a previously unknown tumor-suppressor mechanism that eliminates JNK-activated Drosophila tumors. This mechanism is triggered by the overexpression of microRNAs that downregulate an E3 ubiquitin ligase RNF146, whose loss causes an increase in Tnks (poly-ADP-ribose polymerases) and JNK signaling. This tumor-suppressor mechanism has potential implications for the treatment of JNK-activated tumors. This article is of interest to people in the tumor suppressor, JNK, and miRNA fields, and the key claims are convincing and well supported by the data, and the authors use thoughtful and rigorous approaches.

https://doi.org/10.7554/eLife.77340.sa0

Introduction

Cancer progression is driven by oncogenic alterations of intracellular signaling that lead to promotion of cell proliferation and suppression of cell death (Croce, 2008). The c-Jun N-terminal kinase (JNK) pathway is an evolutionarily conserved mitogen-activated protein (MAP) kinase cascade that regulates both cell proliferation and cell death in normal development and cancer (Bode and Dong, 2007; Eferl and Wagner, 2003). Indeed, JNK signaling can act as both tumor promoter and tumor suppressor depending on the cellular contexts (Bode and Dong, 2007; Bubici and Papa, 2014; Karin and Gallagher, 2005). Crucially, JNK signaling is often activated in various types of cancers (Bubici and Papa, 2014; Wu et al., 2019). Thus, accumulating evidence suggests that JNK signaling can be a critical therapeutic target for cancer. For instance, converting JNK’s role from pro-tumor to antitumor within tumor tissue could be an ideal anticancer strategy.

Drosophila provides a superb model for studying the genetic pathway of cellular signaling and has made great contributions to understand the basic principle of tumor growth and progression (Enomoto et al., 2018; Tipping and Perrimon, 2014). The best-studied model of Drosophila malignant tumor is generated by clones of cells overexpressing oncogenic Ras (RasV12) with simultaneous mutations in apicobasal polarity genes such as lethal giant larvae (lgl), scribble (scrib), or discs large (dlg) in the imaginal epithelium (Brumby and Richardson, 2003; Pagliarini and Xu, 2003). These tumors activate JNK signaling and blocking JNK within the clones strongly suppresses their tumor growth (Igaki et al., 2006; Uhlirova and Bohmann, 2006), indicating that JNK acts as a pro-tumor signaling in these malignant tumors. Conversely, clones of cells overexpressing the oncogene Src in the imaginal discs activate JNK signaling and blocking JNK in these clones results in an enhanced overgrowth (Enomoto and Igaki, 2013), indicating that JNK negatively regulates Src-induced tumor growth. Similarly, although clones of cells mutant for scrib or dlg in the imaginal discs are eliminated by apoptosis when surrounded by wild-type cells, blocking JNK in these clones suppresses elimination and causes tumorous overgrowth (Brumby and Richardson, 2003; Igaki et al., 2009), indicating that JNK acts as antitumor signaling in these mutant clones. Thus, JNK also acts as both pro- and antitumor signaling depending on the cellular contexts in Drosophila imaginal epithelium.

miRNAs are a group of small noncoding RNAs that suppress target gene expression by mRNA degradation or translational repression and have been proposed to be potent targets for cancer therapy. Indeed, several cancer-targeted miRNA drugs have entered clinical trials in recent years. For instance, MRX34, a miRNA mimic drug developed from the tumor-suppressor miR-34a, is the first miRNA-based anticancer drug that has entered phase I clinical trials for patients with advanced solid tumors (Beg et al., 2017; Hong et al., 2020). In addition, MesomiR-1, a miR-16 mimic miRNA that targets EGFR, has entered phase I trial for the treatment of thoracic cancers (Reid et al., 2013; van Zandwijk et al., 2017). Such miRNA-mediated anticancer strategy can be studied using the Drosophila tumor models. Indeed, in Drosophila, the conserved miRNA let-7 targets a transcription factor chinmo and thus suppresses tumor growth caused by polyhomeotic mutations (Jiang et al., 2018). In addition, miR-8 acts as a tumor suppressor against Notch-induced Drosophila tumors by directly inhibiting the Notch ligand Serrate (Vallejo et al., 2011). However, apart from these miRNAs that suppress growth of specific types of tumors, it is unclear whether there exist miRNAs that generally suppress tumor growth caused by different genetic alterations.

Here, using Drosophila tumor models and subsequent genetic analyses, we identified several tumor-suppressor miRNAs. Among these, miR-306 and miR-79, two clustered miRNAs located on the miR-9c/306/79/9b cluster, significantly suppressed growth of multiple types of JNK-activated tumors while showing only a slight effect on normal tissue growth. Mechanistically, miR-306 and miR-79 directly target RNF146, an E3 ubiquitin ligase that causes degradation of a JNK-promoting ADP-ribose polymerase Tnks, thereby overamplifying JNK signaling in tumors to the lethal levels via noncanonical JNK activation. Our findings provide a novel miRNA-based strategy that generally suppress growth of JNK-activating tumors.

Results

Identification of miR-306 and miR-79 as novel tumor-suppressor miRNAs

To identify novel antitumor miRNAs in Drosophila, we focused on 37 miRNA clusters or miRNAs that are highly expressed in Drosophila eye-antennal discs (Chung et al., 2008). Using the Flippase (FLP)-Flp recognition target (FRT)-mediated genetic mosaic technique, each miRNA was overexpressed in clones of cells expressing RasV12 with simultaneous mutations in the apicobasal polarity gene dlg (RasV12/dlg-/-) in the eye-antennal discs, the best-studied malignant tumor model in Drosophila (Pagliarini and Xu, 2003; Figure 1C; compare to Figure 1). We found that overexpression of miR-7, miR-79, miR-252, miR-276a, miR-276b, miR-282, miR-306, miR-310, miR-317, miR-981, miR-988, or the miR-9c/306/79/9b cluster in RasV12/dlg-/- clones dramatically suppressed tumor growth (Figure 1C–G, Figure 1—figure supplement 1D, R, S, T, W, Y, Z, AC, and AE, quantified in Figure 1H and Figure 1—figure supplement 1AI). In addition, overexpression of miR-305, miR-995, or the miR-13a/13b-1/2c cluster mildly suppressed RasV12/dlg-/- tumor growth (Figure 1—figure supplement 1K, X and AF, quantified in Figure 1—figure supplement 1AI). Clustered miRNAs are localized close to each other in the genome and are thus normally transcribed together, ensuring the transcription efficiency of miRNA genes (Kabekkodu et al., 2018; Ryazansky et al., 2011). Notably, overexpression of the miR-9c/306/79/9b cluster, miR-306, or miR-79 dramatically inhibited RasV12/dlg-/- tumor growth (Figure 1E–G, compare to Figure 1D, quantified in Figure 1H). In addition, overexpression of miR-306 or miR-79 was sufficient to rescue the reduced pupation rate and animal lethality caused by RasV12/dlg-/- tumors in the eye-antennal discs (Figure 1I and L). A pervious study in Drosophila wing discs showed that overexpression of miR-79 suppressed tumor growth caused by coexpression of RasV12 and lgl-RNAi via unknown mechanisms (Shu et al., 2017). Similarly, we found that overexpression of the miR-9c/306/79/9b cluster, miR-306, or miR-79 strongly suppressed growth of RasV12/lgl-/- tumors in the eye-antennal discs (Figure 1—figure supplement 2A–D, quantified in Figure 1—figure supplement 2E). Importantly, overexpression of the miR-9c/306/79/9b cluster, miR-306, or miR-79 alone only slightly reduced clone size compared to wild-type (Figure 1M–P, quantified in Figure 1Q). These data indicate that miR-306 and miR-79 are tumor-suppressor miRNAs that only mildly suppress normal tissue growth but specifically block tumor growth in Drosophila imaginal epithelium.

Figure 1 with 2 supplements see all
miR-306 and miR-79 suppress RasV12/dlg-/- tumor growth.

(A–G) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes (A and B, 5 days after egg laying, C–G, 7 days after egg laying). (H) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) in (A–G). Error bars, SD; ****p<0.0001 by one-way ANOVA multiple-comparison test. (I) Pupation rate of flies with indicated genotypes. Data from three independent experiment, n > 30 for each group in one experiment; error bars, SD. (J) Eclosion rate of flies with indicated genotypes. Data from three independent experiment, n > 30 for each group in one experiment; error bars, SD. (K, L) Adult eye phenotype of flies with indicated genotypes. (M–P) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes (5 days after egg laying). (Q) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) of (M–P). Error bars, SD; **p<0.01, ****,p<0.0001 by one-way ANOVA multiple-comparison test.

miR-306 and miR-79 suppress tumor growth by promoting cell death

We next investigated the mechanism by which miR-306 and miR-79 suppress tumor growth. Immunostaining of RasV12/dlg-/- or RasV12/lgl-/- tumors with anti-cleaved DCP-1 antibody revealed that expression of the miR-9c/306/79/9b cluster, miR-306, or miR-79 in tumor clones significantly increased the number of dying cells (Figure 2A–E, Figure 2—figure supplement 1A–D, quantified in Figure 2F and Figure 2—figure supplement 1E). In addition, blocking cell death in tumor clones by overexpressing the caspase inhibitor baculovirus p35 canceled the tumor-suppressive activity of miR-306 or miR-79, while p35 overexpression alone did not affect growth of normal tissues or RasV12/dlg-/- tumors (Figure 2G–N, quantified in Figure 2O). These data indicate that the miR-9c/306/79/9b cluster, miR-306, or miR-79 suppresses tumor growth by inducing cell death. Importantly, overexpression of these miRNAs alone did not cause cell death in normal tissue (Figure 2P–S, quantified in Figure 2T), suggesting that miR-306 or miR-79 cooperates with a putative tumor-specific signaling activated in RasV12/dlg-/- or RasV12/lgl-/- tumors to induce synthetic lethality.

Figure 2 with 1 supplement see all
miR-306 and mir-79 suppress RasV12/dlg-/- tumor growth by inducing apoptosis.

(A–E) Eye-antennal disc bearing GFP-labeled clones (A’-E’) of indicated genotypes stained with anti-cleaved Dcp-1 antibody (A-E and A’-E’, A, 5 days after egg laying, B–E, 7 days after egg laying). (F) Quantification of dying cells in GFP-positive clone area in (A–E). Error bars, SD; **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA multiple-comparison test. (G–N) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes (G and H, 5 days after egg laying, I–N, 7 days after egg laying). (O) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) of (G–N). Error bars, SD; n.s., p>0.05 (not significant), ****p<0.0001 by one-way ANOVA multiple-comparison test. (P–S) Eye-antennal disc bearing GFP-labeled clones (P’-S’) of indicated genotypes stained with anti-cleaved Dcp-1 antibody (P-S and P’-S’, 5 days after egg laying). (T) Quantification of dying cells in GFP-positive clone area in (P–S). Error bars, SD; n.s., p>0.05 (not significant) by one-way ANOVA multiple-comparison test.

miR-306 and miR-79 suppress tumor growth by enhancing JNK signaling

We thus examined whether Ras activation or cell polarity defect cooperates with miR-306 or miR-79 to induce cell death. Overexpression of the miR-9c/306/79/9b cluster, miR-306, or miR-79 in RasV12-expresing clones did not affect their growth (Figure 3—figure supplement 1A–D, quantified in Figure 3—figure supplement 1E), indicating that Ras signaling does not cooperate with these miRNAs. Notably, overexpression of these miRNAs in dlg-/- clones significantly reduced their clone size (Figure 3A–E, quantified in Figure 3F). In addition, blocking cell death by overexpression of p35 canceled the ability of these miRNAs to reduce dlg-/- clone size (Figure 3G–O, quantified in Figure 3P), suggesting that these miRNAs block dlg-/- clone growth by promoting cell death. These data show that miR-306 or miR-79 cooperates with loss of cell polarity to induce synthetic lethality.

Figure 3 with 3 supplements see all
miR-306 and mir-79 suppress tumor growth and promote cell competition by promoting JNK signaling.

(A–E) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes (5 days after egg laying). (F) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) of (A–E). Error bars, SD; ****p<0.0001 by one-way ANOVA multiple-comparison test. (G–O) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes (5 days after egg laying). (P) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) of (A, G–O). Error bars, SD; n.s., p>0.05 (not significant), **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA multiple-comparison test. (Q–W) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes (Q, 5 days after egg laying, R–W, 7 days after egg laying). (X) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) of (A, Q–W). Error bars, SD; n.s., p>0.05 (not significant), **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA multiple-comparison test. (Y–AA) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes (5 days after egg laying). (AB) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) of (H, J, K, Y–AA). Error bars, SD; ****p<0.0001 by one-way ANOVA multiple-comparison test.

We then sought to identify the polarity defect-induced intracellular signaling that cooperates with miR-306 or miR-79 to induce cell death. It has been shown that clones of cells mutant for cell polarity genes such as dlg activate JNK signaling via the Drosophila tumor necrosis factor (TNF) Eiger (Brumby and Richardson, 2003; Igaki et al., 2009). We found that overexpression of miR-306 or miR-79 alone moderately activated JNK signaling in the eye-antennal discs, as visualized by anti-p-JNK antibody staining and the puc-LacZ reporter (Figure 3—figure supplement 2A–F). In addition, Western blot analysis with anti-p-JNK antibody revealed that overexpression of miR-306 or miR-79 in the eyes using the GMR-Gal4 driver caused JNK activation (Figure 3—figure supplement 2G, quantified in Figure 3—figure supplement 2H). Notably, although overexpression of miR-306 or miR-79 alone in the eyes had no significant effect on eye morphology (Figure 3—figure supplement 2I–K, quantified in Figure 3—figure supplement 2L), they dramatically enhanced the reduced-eye phenotype caused by overexpression of Eiger (Figure 3—figure supplement 2M–O, quantified in Figure 3—figure supplement 2P). It has been shown that the severity of the reduced-eye phenotype depends on the levels of JNK activation and subsequent cell death (Igaki et al., 2002; Igaki et al., 2006; Igaki et al., 2009), suggesting that miR-306 and miR-79 enhance Eiger-mediated activation of JNK signaling. Indeed, blocking JNK signaling by overexpression of a dominant-negative form of Drosophila JNK Basket (BskDN) canceled the tumor-suppressive activity of miR-306 or miR-79 against RasV12/dlg-/- or RasV12/lgl-/- tumors (Figure 3R–W, quantified in Figure 3X, and Figure 3—figure supplement 3A–C, quantified in Figure 3—figure supplement 3D), while BskDN did not affect growth of normal tissues (Figure 3Q compare to Figure 3A, quantified in Figure 3X). Moreover, overexpression of BskDN significantly increased the size of dlg-/- or lgl-/- clones overexpressing miR-306 or miR-79 (Figure 3H, J, K, Y, Z, and AA, quantified in Figure 3AB; Figure 3—figure supplement 3E–J, quantified in Figure 3—figure supplement 3K). Together, these data suggest that miR-306 and miR-79 suppress growth of malignant tumors by enhancing JNK signaling activation.

miR-306 and miR-79 enhance JNK signaling stimulated by different upstream signaling

We next examined whether miR-306 or miR-79 suppresses growth of other types of tumors with elevated JNK signaling via an Eiger-independent mechanism. Overexpression of an activated form of the Drosophila PDGF/VEGF receptor homolog (PVRact) results in JNK activation and tumor formation in the wing disc (Wang et al., 2016a) and eye-antennal disc (Figure 4B, compare to Figure 4A, quantified in Figure 4F). This tumor growth was significantly suppressed by overexpression of the miR-9c/306/79/9b cluster, miR-306, or miR-79 (Figure 4B–E, quantified in Figure 4F). In addition, the size of clones overexpressing the oncogene Src64B in the eye-antennal disc (Figure 4H, compare to Figure 4G, quantified in Figure 4F), which activate JNK signaling (Enomoto and Igaki, 2013), was significantly reduced when the miR-9c/306/79/9b cluster, miR-306, or miR-79 was coexpressed (Figure 4H–K, quantified in Figure 4L). Moreover, nonautonomous overgrowth of surrounding wild-type tissue by Src64B-overexpressing clones (Enomoto and Igaki, 2013) was significantly suppressed by coexpression of these miRNAs (Figure 4M–P, quantified in Figure 4Q). Furthermore, the size of clones mutant for an RNA helicase Hel25E or an adaptor protein Mahj, both of which are eliminated by JNK-dependent cell competition when surrounded by wild-type cells (Nagata et al., 2019; Tamori et al., 2010), was significantly reduced when these miRNAs were coexpressed (Figure 4—figure supplement 1A–D, quantified in Figure 4—figure supplement 1E, and Figure 4—figure supplement 1F–I, quantified in Figure 4—figure supplement 1J). In these tumors or cell competition models, ectopic expression of miR-306 or miR-79 enhanced JNK activity (Figure 4—figure supplement 2). We further examined whether expression of these miRNAs enhances normally occurring JNK activity during development. The pnr-GAL4 driver strain specifically expresses GAL4 in the wing discs in a broad domain corresponding to the central presumptive notum during metamorphosis (Ishimaru et al., 2004; Zeitlinger and Bohmann, 1999). Knocking down Hep, the Drosophila JNK kinase, using the pnr-GAL4 driver generates a split-thorax phenotype caused by reduced JNK signaling (Ishimaru et al., 2004). On the contrary, ectopic expression of Hep or Eiger using pnr-GAL4 generates a small-scutellum phenotype caused by elevated JNK signaling (Ma et al., 2013; Xue et al., 2007). Similarly to Hep or Eiger, ectopic expression of miR-306 or miR-79 using pnr-GAL4 resulted in a small-scutellum phenotype (Figure 4—figure supplement 3A–C, quantified in Figure 4—figure supplement 3D). These data suggest that miR-306 and miR-79 broadly enhance JNK signaling activity stimulated by different upstream signaling.

Figure 4 with 3 supplements see all
miR-306 and miR-79 suppress growth of multiple types of tumor models.

(A–E) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes (A, 5 days after egg laying, B–E, 7 days after egg laying). (F) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) of (A–E). Error bars, SD; ****p<0.0001 by one-way ANOVA multiple-comparison test. (G–K) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes (G, 5 days after egg laying, H–K, 6 days after egg laying). (L) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) of (G–K). Error bars, SD; ****p<0.0001 by one-way ANOVA multiple-comparison test. (M–P) Adult eye phenotype of flies with indicated genotypes. (Q) Quantification of percentage of folded eye in (M–P). n = 20 for each group.

miR-306 and miR-79 enhance JNK signaling activity by targeting RNF146

We next sought to identify the mechanism by which miR-306 and miR-79 enhance JNK signaling by searching for the target gene(s) of these miRNAs. The clustered miRNAs often target overlapping sets of genes and thus co-regulate various biological processes (Kim et al., 2009; Wang et al., 2016b; Yuan et al., 2009). Given that miR-306 and miR-79 are located on the same miRNA cluster, we searched for the common targets of these miRNAs using the online software TargeyScanFly 7.2 (http://www.targetscan.org/fly_72/) and found 11 mRNAs that were predicted to be targets of both miR-306 and miR-79 (Figure 5A). We then examined whether knocking down of each one of these candidate genes could activate JNK signaling in Drosophila wing discs, where a clear JNK activation was observed when miR-306 or miR-79 was overexpressed (Figure 5—figure supplement 1). As a result, we found that knocking down of RNF146, but not any other available RNAis for the candidate genes, resulted in JNK activation (Figure 5B and C, Figure 5—figure supplement 2). The RNF146 mRNA had putative target sites of miR-306 and miR-79 in its 3′UTR region (Figure 5D). To confirm that RNF146 mRNA is a direct target of miR-306 and miR-79, we performed a dual-luciferase reporter assay in Drosophila S2 cells using wild-type RNF146 3′UTR (RNF146 WT) or mutant RNF146 3′UTR bearing mutations at the putative binding site of miR-306 (RNF146 m1) or miR-79 (RNF146 m2) (Figure 5D). We found that miR-306 and miR-79 reduced wild-type RNF146 3′UTR expression but did not affect respective mutant RNF146 3′UTR (Figure 5E), indicating that miR-306 and miR-79 directly target RNF146 3′UTR (Figure 5D). We also confirmed that overexpression of miR-306 or miR-79 reduced the endogenous levels of RNF146 protein (Figure 5F, quantified in Figure 5G) and that suppression of miR-306 and miR-79 functions by using miRNA sponges increased the endogenous levels of RNF146 protein in the adult eyes (Figure 5—figure supplement 3A, quantified in Figure 5—figure supplement 3B).

Figure 5 with 6 supplements see all
miR-306 and mir-79 suppress tumor growth and promote cell competition by targeting RNF146.

Predicted targets of miR-306 and miR-79. (B, C) Wing disc of indicated genotypes with puc-lacZ background stained with anti-β-galactosidase antibody (B,C and B’, C’, 5 or 6 days after egg laying). (D) Schematic of the wild-type and mutation-type 3′UTR vector with miRNA binding sites for miR-306 and miR-79, respectively. Red letters shows the mutation sites. Red box shows the seed sequence pairing region. (E) RLU/FLU rate from dual-luciferase assay. n = 3, error bars, SD; n.s., p>0.05 (not significant), **p<0.01 by two-tailed Student’s t-test. (F) Lysates of adult heads of indicated genotypes were subjected to Western blots using indicated antibodies. (G) Quantification of relative levels of RNF146 protein in (F) from three independent experiments. Error bars, SD; *p<0.05, **p<0.01 by one-way ANOVA multiple-comparison test. (H–J) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes (7 days after egg laying). (K) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) of (H–J). Error bars, SD; ****p<0.0001 by two-tailed Student’s t-test. (L–M) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes (7 days after egg laying). (N) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) of (L–M). Error bars, SD; n.s., p>0.05 (not significant) by two-tailed Student’s t-test. (O–R) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes (7 days after egg laying). (S) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) of (O–R). Error bars, SD; *p<0.05, **p<0.01 by one-way ANOVA multiple-comparison test.

We next investigated whether RNF146 is the responsible target of miR-306 and miR-79 for the enhancement of JNK signaling. We found that, while knockdown of RNF146 did not affect normal tissue growth (Figure 5—figure supplement 4A and B, quantified in Figure 5—figure supplement 4C), it significantly suppressed RasV12/dlg-/- tumor growth (Figure 5H–J, quantified in Figure 5K) and promoted elimination of dlg-/- clones (Figure 5—figure supplement 4D and E, quantified in Figure 5—figure supplement 4F). Although overexpression of one copy of miR-306 or miR-79 in the eyes had no significant effect on the number of dying cells or eye morphology (Figure 3—figure supplement 2I–K, quantified in Figure 3—figure supplement 2L, Figure 5—figure supplement 4G–J, quantified in Figure 5—figure supplement 4N), overexpression of 2×miR-306 or 2×miR-79 in the eyes significantly increased the number of dying cells (Figure 5—figure supplement 4K–M, quantified in Figure 5—figure supplement 4N) and resulted in a reduced-eye phenotype (Figure 5—figure supplement 4O and Q). Overexpression of RNF146 rescued the reduced-eye phenotype caused by overexpression of 2×miR-306 or 2×miR-79 in the eyes (Figure 5—figure supplement 4O–R, quantified in Figure 5—figure supplement 4S). Moreover, knocking down of RNF146 significantly enhanced Eiger-induced reduced-eye phenotype (Figure 5—figure supplement 4T–W, quantified in Figure 5—figure supplement 4X). Furthermore, although overexpression of RNF146 did not affect RasV12/dlg-/- tumor growth (Figure 5L–M, quantified in Figure 5N), overexpression of RNF146 weakened the tumor-suppressive effect of miR-306 or miR-79 on RasV12/dlg-/- tumors (Figure 5O–R, quantified in Figure 5S). The RNF146 overexpression also weakened the enhanced elimination of dlg-/- clones by miR-306 or miR-79 (Figure 5—figure supplement 4Y–AB, quantified in Figure 5—figure supplement 4AC). Similarly to ectopic expression of miR-306 or miR-79, knocking down of RNF146 enhanced JNK activity in RasV12/dlg-/- tumors (Figure 5—figure supplement 5A and B, quantified in Figure 5—figure supplement 5C). Together, these data indicate that miR-306 and miR-79 directly target RNF146 mRNA, thereby enhancing JNK signaling activity and thus exerting the tumor-suppressive effects.

RNF146 promotes Tnks degradation

We next investigated the mechanism by which downregulation of RNF146 by miR-306 or miR-79 enhances JNK signaling activity. It has been shown in Drosophila embryos, larvae, wing discs, and adult eyes that loss of RNF146 upregulates the protein levels of Tnks (Gultekin and Steller, 2019; Wang et al., 2019), a poly-ADP-ribose polymerase that directly mediates poly-ADP ribosylation of JNK, which triggers K63-linked poly-ubiquitination of JNK and thereby promotes JNK-dependent apoptosis in Drosophila (Feng et al., 2018; Li et al., 2018). In addition, loss of RNF146 was shown to enhance rough-eye phenotype caused by Tnks overexpression (Gultekin and Steller, 2019). These observations raise the possibility that downregulation of RNF146 by miR-306 or miR-79 enhances JNK signaling via upregulation of Tnks. Indeed, as reported previously (Feng et al., 2018; Li et al., 2018), Western blot analysis revealed that overexpression of Tnks induces phosphorylation of JNK (JNK activation) in S2 cells (Figure 6A, lane 2 vs. lane 1, quantified in Figure 6C). Notably, coexpression of RNF146 significantly downregulated Tnks protein level and suppressed Tnks-induced JNK phosphorylation (Figure 6A, lane 3 vs. lane 2, quantified in Figure 6B and C). Moreover, knocking down of RNF146 or overexpression of miR-306 or miR-79 significantly upregulated Tnks protein level and promoted JNK phosphorylation (Figure 6D, quantified in Figure 6E and F, Figure 6—figure supplement 1A, quantified in Figure 6—figure supplement 1B and C). These data support the notion that downregulation of RNF146 or overexpression of miR-306 or miR-79 enhances JNK activation via upregulation of Tnks. Indeed, overexpression of Tnks was sufficient to suppress growth of either normal tissues or RasV12/dlg-/- tumors (Figure 6G–J, quantified in Figure 6K). Due to the fact that overexpression of Tnks alone resulted in larger clone size than RasV12/dlg-/-+Tnks clone (Figure 6H and J, quantified in Figure 6K), our data support the notion that Tnks suppresses growth of RasV12/dlg-/- tumors by cooperating with JNK signaling. Consistent with the data shown above, overexpression of Tnks rescued the lethality of flies bearing RasV12/dlg-/- tumors in the eye-antennal discs (Figure 6L and M).

Figure 6 with 1 supplement see all
RNF146 promotes poly-ubiquitination and degradation of Tnks.

(A) Drosophila S2 cells were transfected with plasmids expressing indicated proteins. Cell lysates were subjected to Western blots using indicated antibodies. (B) Quantification of relative Tnks-myc levels in (A) from three independent experiments. Error bars, SD; ***p<0.001 by two-tailed Student’s t-test. (C) Quantification of relative p-JNK levels in (A) from three independent experiments. Error bars, SD; *p<0.05 by one-way ANOVA multiple-comparison test. (D) Drosophila S2 cells were transfected with plasmid expressing indicated protein and dsRNA targeting indicated gene. (E, F) Quantification of relative Tnks-myc levels (E) and p-JNK (F) levels in (D) from three independent experiments. Error bars, SD; *p<0.05, ***p<0.001 by one-way ANOVA multiple-comparison test. (G–J) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes (G, H, 5 days after egg laying, I, J, 7 days after egg laying). (K) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) of (G–J). Error bars, SD; ****p<0.0001 by one-way ANOVA multiple-comparison test. (L) Adult eye phenotype of flies with indicated genotypes. (M) Eclosion rate of flies with indicated genotypes. Data from three independent experiment, n > 30 for each group in one experiment; error bars, SD. (N, O) Drosophila S2 cells were transfected with plasmid expressing indicated protein and dsRNA targeting indicated gene. After 36 hr, cells were treated with 50 μg/ml cycloheximide (CHX) for the indicated periods. Cell lysates were subjected to Western blots using indicated antibodies. (P) Quantification of relative Tnks-myc levels in (N, O) from three independent experiments. Error bars, SD. (Q) A model for tumor elimination by miR-306/79. Tumor cell with elevated canonical JNK signaling via Eiger/TNF, dTAK1/JNKKK, and Hep/JNKK grows in a Bsk/JNK-dependent manner. Overexpression of miR-306 or miR-79 in JNK-activated tumor cell results in overactivation of JNK signaling to the lethal level via RNF146-Tnks-mediated noncanonical JNK-activating signaling. Overexpression of miR-306 or miR-79 in normal cells has no significant effect on JNK signaling.

Finally, we sought to clarify the mechanism by which downregulation of RNF146 upregulates Tnks. A pervious study has shown that Tnks protein levels were significantly higher in Rnf146 mutant background than in wild-type (Gultekin and Steller, 2019). However, this upregulation of Tnks can be caused by either elevated Tnks protein synthesis or reduced Tnks protein degradation. We thus examined the possibility that RNF146 promotes degradation of Tnks. Blocking new protein synthesis in S2 cells by the protein synthesis inhibitor cycloheximide (CHX) resulted in a time-dependent depletion of Tnks protein with a half-life of less than 3 hr (Figure 6N, quantified in Figure 6P). This depletion of Tnks was significantly retarded when RNF146 was knocked down (Figure 6O, quantified in Figure 6P). These data indicate that endogenous RNF146 promotes degradation of Tnks protein. Taken together, our data show that miR-306 or miR-79 directly targets RNF146, thereby leading to elevation of Tnks protein that induces noncanonical activation of JNK signaling (Figure 6Q).

Discussion

In this study, we have identified the clustered miRNAs miR-306 and miR-79 as novel antitumor miRNAs that selectively eliminate JNK-activated tumors from Drosophila imaginal epithelia. Mechanistically, miR-306 and miR-79 directly target RNF146, an E3 ligase that promotes degradation of a poly-ADP-ribose polymerase Tnks, thereby leading to upregulation of Tnks and thus promoting JNK activation (Figure 6K). Importantly, this noncanonical mode of JNK activation has only a weak effect on normal tissue growth but it strongly blocks tumor growth by overactivating JNK signaling when tumors already possess elevated JNK signaling via the canonical JNK pathway (Figure 6K). Given that tumors or premalignant mutant cells often activate canonical JNK signaling, miR-306 and miR-79 can be novel ideal targets of cancer therapy.

Our study identified several putative co-target genes of miR-306 and miR-79 (Figure 5A). Interestingly, some of these genes (Atf3, chinmo, and chn) have been reported to be involved in tumor growth in Drosophila. Atf3 encodes an AP-1 transcription factor that was shown to be a polarity-loss responsive gene acting downstream of the membrane-associated Scrib polarity complex (Donohoe et al., 2018). Knockdown of Atf3 suppresses growth and invasion of RasV12/scrib-/- tumors in eye-antennal discs (Atkins et al., 2016). Chinmo is a BTB-zinc finger oncogene that is upregulated by JNK signaling in tumors (Doggett et al., 2015). Although loss of chinmo does not significantly suppress tumor growth, overexpression of chinmo with RasV12 or an activated Notch is sufficient to promote tumor growth in eye-antennal discs (Doggett et al., 2015). Chn encodes a zinc finger transcription factor that cooperates with scrib-/- to promote tumor growth (Turkel et al., 2013). Although we found that knockdown of these genes did not activate JNK signaling, it is possible that these putative target genes also contribute to the miR-306/miR-79-induced tumor suppression.

Intriguingly, it has been reported that miR-79 is downregulated in RasV12/lgl-RNAi tumors in Drosophila wing discs (Shu et al., 2017). Given that miR-306 is located in the same miRNA cluster with miR-79, it is highly possible that miR-306 is also downregulated in tumors. This suggests that tumors have the mechanism that downregulates antitumor miRNAs for their survival and growth. Future studies on the mechanism of how tumors regulate these miRNAs would provide new understanding of tumor biology.

Our study uncovered the miR-306/79-RNF146-Tnks axis as noncanonical JNK enhancer that selectively eliminates JNK-activated tumors in Drosophila. Considering that miR-9, the mammalian homolog of miR-79, is predicted to target mammalian RNF146 (Figure 5—figure supplement 6) and that JNK signaling is highly conserved throughout evolution, it opens up the possibility of developing a new miRNA-based strategy against cancer.

Materials and methods

Fly stocks

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All flies used were reared at 25°C on a standard cornmeal/yeast diet. Fluorescently labeled mitotic clones were produced in larval imaginal discs using the following strains: Tub-Gal80, FRT40A; eyFLP6, Act>y+>Gal4, UAS-GFP (40A tester), FRT42D, Tub-Gal80/CyO; eyFLP6, Act>y+>Gal4, UAS-GFP (42D tester), Tub-Gal80, FRT19A; eyFLP5, Act>y+>Gal4, UAS-GFP (19A tester #1), Tub-Gal80, FRT19A; eyFLP6, Act>y+>Gal4, UAS-GFP (19A tester #2). Additional strains used are the following: dlgm52 (Goode and Perrimon, 1997), puc-lacZ (Igaki et al., 2006), UAS-Rasv12 (Igaki et al., 2006), UAS-BskDN (Adachi-Yamada et al., 1999), UAS-Src64B (Wills et al., 1999), Hel25Eccp-8 (Nagata et al., 2019), Mahj1 (Tamori et al., 2010), UAS-Nact (Hori et al., 2004), UAS-RNF146 (Gultekin and Steller, 2019); lgl4 (BDSC #36289), UAS-p35 (BDSC #5073), UAS-PVRact (BDSC #58496), UAS-YkiS168A (BDSC #28836), UAS-Luciferase (BDSC #35788), UAS-RFP (BDSC #30556), UAS-bantam (BDSC #60672), UAS-miR-9c,306,79,9b (BDSC #41156), UAS-miR-79 (BDSC #41145), UAS-miR-2a-2,2a-1,2b-2 (BDSC #59849), UAS-miR-2b-1 (BDSC #41128), UAS-miR-7 (BDSC #41137), UAS-miR-8 (BDSC #41176), UAS-miR-9a (BDSC #41138), UAS-miR-9b (BDSC #41131), UAS-miR-9c (BDSC #41139), UAS-miR-11 (BDSC #59865), UAS-miR-12 (BDSC #41140), UAS-miR-13a,13b-1,2c (BDSC #64097), UAS-miR-13b-2 (BDSC #59867), UAS-miR-14 (BDSC #41178), UAS-miR-34 (BDSC #41158), UAS-miR-92a (BDSC #41153), UAS-miR-124 (BDSC #41126), UAS-miR-184 (BDSC #41174), UAS-miR-252 (BDSC #41127), UAS-miR-276a (BDSC #41143), UAS-miR-276b (BDSC #41162), UAS-miR-278 (BDSC #41180), UAS-miR-279 (BDSC #41147), UAS-miR-282 (BDSC #41165), UAS-miR-305 (BDSC #41152), UAS-miR-310 (BDSC #41155), UAS-miR-317 (BDSC #59913), UAS-miR-958 (BDSC #41222), UAS-miR-975,976,977 (BDSC #60635), UAS-miR-981 (BDSC #60639), UAS-miR-984 (BDSC #41224), UAS-miR-988 (BDSC #41196), UAS-miR-995 (BDSC #41199), UAS-miR-996 (BDSC #60653), UAS-miR-998 (BDSC #63043), UAS-miR-306-sponge (BDSC #61424), UAS-miR-79-sponge (BDSC #61387), UAS-Luciferase RNAi (BDSC #31603), UAS-aop RNAi (BDSC #34909), UAS-pde1c RNAi (BDSC #55925), UAS-atf3 RNAi (BDSC #26741), UAS-mei-P26 RNAi (BDSC #57268), UAS-chn RNAi (BDSC #26779), UAS-chinmo RNAi (BDSC #26777), UAS-RNF146 RNAi (BDSC #40882), UAS-bcd RNAi (BDSC #33886) and UAS-CG1358 RNAi (BDSC #64848) from Bloomington Drosophila Stock Center; UAS-miR-306 (FlyORF #F002214) from FlyORF; UAS-Tnks from Core Facility of Drosophila Resource and Technology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences.

Clone size measurement

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Eye-antennal disc images were taken with a Leica SP8 confocal microscope or Olympus Fluoview FV3000 confocal microscope. To measure clone size, ImageJ (Fiji) software was used to determine the threshold of the fluorescence. Total clone area/disc area (%) in the eye-antennal disc was calculated using ImageJ and Prism 8 (GraphPad).

Histology

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Larval tissues were stained with standard immunohistochemical procedures using rabbit anti-phospho-JNK polyclonal antibody (Cell Signaling Technology, Cat #4668, 1:100), chicken anti-β-galactosidase antibody (Abcam, Cat #ab9361, 1:1000), rabbit anti-Cleaved Drosophila Dcp-1 (Asp216) antibody (Cell Signaling Technology, Cat #9578, 1:100), goat anti-rabbit secondary antibody, Alexa Fluor 647 (Thermo Fisher Scientific, Cat #A32733, 1:250) or goat anti-chicken secondary antibody, Alexa Fluor 647 (Thermo Fisher Scientific, Cat #A21449, 1:250). Samples were mounted with DAPI-containing SlowFade Gold Antifade Reagent (Thermo Fisher Scientific, Cat #S36937). Images were taken with a Leica SP8 confocal microscope. The cleaved Dcp-1 positive cell number and the P-JNK-positive area was calculated using ImageJ and Prism 8 (GraphPad).

Plasmid and in vitro transcription of dsRNA

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pAc5.1/V5-His vector (Thermo Fisher Scientific, Cat #V411020) was used to construct plasmids for expressing proteins or miRNAs in Drosophila S2 cells. The RNF146 or Tnks ORF was amplified from fly cDNAs via PCR. The RNF146 ORF was cloned into the EcoR І-Xho І site of the pAc5.1/V5-His vector. The Tnks ORF carrying a myc tag at its 5′-end was cloned into the Kpn І-Xho І site of the pAc5.1/V5-His vector. Extended region of miR-306 (–184 to +136) or miR-79 (–124 to +131) was amplified from fly cDNAs via PCR and cloned into the Kpn І-EcoR І site of the pAc5.1/V5-His vector.

RNF146 dsRNA #1 and #2, respectively, targeting the 1–318 and 319–667 region of RNF146 ORF, used for RNF146 RNAi were transcribed in vitro using T7 RNA polymerase (Promega, Cat #P2075) at 37°C for 4 hr from the PCR products.

Cell culture and transfection

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Drosophila S2-ATCC cells (RRID:CVCL_Z232) was obtained from American Type Culture Collection (ATCC). Its identity was confirmed by visual inspection of the cell morphology and its growth kinetics in Schneider’s Drosophila medium (Thermo Fisher Scientific, Cat #21720024)/10% fetal bovine serum (FBS) and penicillin/streptomycin. A mycoplasma test is usually not done for S2 cells.

For transfection assay, S2 cells were plated in 100 mm plates or six-well plates and grown overnight to reach 70% confluence. After that, DNA plasmids or dsRNAs were transfected into the cells using FuGene HD transfection reagent (Promega, Cat #PRE2311) according to the manufacturer’s protocol. The protein synthesis inhibitor CHX (Santa Cruz Biotechnology, Cat #SC-3508) was used at 50 μg/ml.

Western blots

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Cultured Drosophila S2 cells were harvested and then lysed in cell lysis buffer. The cell lysates were then subjected to SDS-PAGE, followed by Western blots using anti-α-tubulin monoclonal antibody (Sigma-Aldrich, Cat #T5168, 1:5000), anti-phospho-JNK polyclonal antibody (Cell Signaling Technology, Cat #9251, 1:1000), anti-JNK monoclonal antibody (Santa Cruz Biotechnology, Cat #sc-7345, 1:1000), anti-RNF146 polyclonal antibody (raised in rabbits against the peptide HSGGGSGEDPAVGSC, GenScript antibody service, Nanjing, China, 1:2000), anti-V5 tag monoclonal antibody (Thermo Fisher Scientific, Cat #R960-25, 1:5000), anti-myc tag polyclonal antibody (MBL, Code #562, 1:1000), anti-mouse IgG, HRP-linked antibody (Cell Signaling Technology, Cat #7076, 1:5000), or anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, Cat #7074, 1:5000).

Dual-luciferase reporter assay

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The psiCHECK-2 vector (Promega, Cat #C8021) was used to construct plasmids for dual-luciferase reporter assay. RNF146 3′UTR or its mutant was cloned into the Xho І-Not I site of the psiCHECK-2 vector. Renilla luciferase activity and firefly luciferase activity were measured using GloMax-Multi Jr Single-Tube Multimode Reader (Promega) according to the manufacturer’s protocol.

Statistical analysis

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When comparing two groups, statistical significance was tested using a Student’s t-test. When comparing multiple groups, statistical significance was tested using a one-way ANOVA multiple-comparison test. In all figures, significance is indicated as follows: n.s. (not significant), p>0.05, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Appendix 1

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Drosophila melanogaster)dlgm52PMID:9334318N/A
Genetic reagent (D. melanogaster)puc-lacZPMID:16753569N/A
Genetic reagent (D. melanogaster)UAS-Rasv12PMID:16753569N/A
Genetic reagent (D. melanogaster)UAS-BskDNPMID:10490662N/A
Genetic reagent (D. melanogaster)UAS-Src64BPMID:10069336N/A
Genetic reagent (D. melanogaster)Hel25Eccp-8PMID:31543447N/A
Genetic reagent (D. melanogaster)Mahj1PMID:20644714N/A
Genetic reagent (D. melanogaster)UAS-NactPMID:15496440N/A
Genetic reagent (D. melanogaster)UAS-RNF146PMID:30796047N/A
Genetic reagent (D. melanogaster)lgl4Bloomington Drosophila Stock CenterBDSC:36289
Genetic reagent (D. melanogaster)UAS-p35Bloomington Drosophila Stock CenterBDSC:5073
Genetic reagent (D. melanogaster)UAS-PVRactBloomington Drosophila Stock CenterBDSC:58496
Genetic reagent (D. melanogaster)UAS-YkiS168ABloomington Drosophila Stock CenterBDSC:28836
Genetic reagent (D. melanogaster)UAS-LuciferaseBloomington Drosophila Stock CenterBDSC:35788
Genetic reagent (D. melanogaster)UAS-RFPBloomington Drosophila Stock CenterBDSC:30556
Genetic reagent (D. melanogaster)UAS-bantamBloomington Drosophila Stock CenterBDSC:60672
Genetic reagent (D. melanogaster)UAS-miR-9c,306,79,9bBloomington Drosophila Stock CenterBDSC:41156
Genetic reagent (D. melanogaster)UAS-miR-79Bloomington Drosophila Stock CenterBDSC:41145
Genetic reagent (D. melanogaster)UAS-miR-2a-2,2a-1,2b-2Bloomington Drosophila Stock CenterBDSC:59849
Genetic reagent (D. melanogaster)UAS-miR-2b-1Bloomington Drosophila Stock CenterBDSC:41128
Genetic reagent (D. melanogaster)UAS-miR-7Bloomington Drosophila Stock CenterBDSC:41137
Genetic reagent (D. melanogaster)UAS-miR-8Bloomington Drosophila Stock CenterBDSC:41176
Genetic reagent (D. melanogaster)UAS-miR-9aBloomington Drosophila Stock CenterBDSC:41138
Genetic reagent (D. melanogaster)UAS-miR-9bBloomington Drosophila Stock CenterBDSC:41131
Genetic reagent (D. melanogaster)UAS-miR-9cBloomington Drosophila Stock CenterBDSC:41139
Genetic reagent (D. melanogaster)UAS-miR-11Bloomington Drosophila Stock CenterBDSC:59865
Genetic reagent (D. melanogaster)UAS-miR-12Bloomington Drosophila Stock CenterBDSC:41140
Genetic reagent (D. melanogaster)UAS-miR-13a,13b-1,2cBloomington Drosophila Stock CenterBDSC:64097
Genetic reagent (D. melanogaster)UAS-miR-13b-2Bloomington Drosophila Stock CenterBDSC:59867
Genetic reagent (D. melanogaster)UAS-miR-14Bloomington Drosophila Stock CenterBDSC:41178
Genetic reagent (D. melanogaster)UAS-miR-34Bloomington Drosophila Stock CenterBDSC:41158
Genetic reagent (D. melanogaster)UAS-miR-92aBloomington Drosophila Stock CenterBDSC:41153
Genetic reagent (D. melanogaster)UAS-miR-124Bloomington Drosophila Stock CenterBDSC:41126
Genetic reagent (D. melanogaster)UAS-miR-184Bloomington Drosophila Stock CenterBDSC:41174
Genetic reagent (D. melanogaster)UAS-miR-252Bloomington Drosophila Stock CenterBDSC:41127
Genetic reagent (D. melanogaster)UAS-miR-276aBloomington Drosophila Stock CenterBDSC:41143
Genetic reagent (D. melanogaster)UAS-miR-276bBloomington Drosophila Stock CenterBDSC:41162
Genetic reagent (D. melanogaster)UAS-miR-278Bloomington Drosophila Stock CenterBDSC:41180
Genetic reagent (D. melanogaster)UAS-miR-279Bloomington Drosophila Stock CenterBDSC:41147
Genetic reagent (D. melanogaster)UAS-miR-282Bloomington Drosophila Stock CenterBDSC:41165
Genetic reagent (D. melanogaster)UAS-miR-305Bloomington Drosophila Stock CenterBDSC:41152
Genetic reagent (D. melanogaster)UAS-miR-310Bloomington Drosophila Stock CenterBDSC:41155
Genetic reagent (D. melanogaster)UAS-miR-317Bloomington Drosophila Stock CenterBDSC:59913
Genetic reagent (D. melanogaster)UAS-miR-958Bloomington Drosophila Stock CenterBDSC:41222
Genetic reagent (D. melanogaster)UAS-miR-975,976,977Bloomington Drosophila Stock CenterBDSC:60635
Genetic reagent (D. melanogaster)UAS-miR-981Bloomington Drosophila Stock CenterBDSC:60639
Genetic reagent (D. melanogaster)UAS-miR-984Bloomington Drosophila Stock CenterBDSC:41224
Genetic reagent (D. melanogaster)UAS-miR-988Bloomington Drosophila Stock CenterBDSC:41196
Genetic reagent (D. melanogaster)UAS-miR-995Bloomington Drosophila Stock CenterBDSC:41199
Genetic reagent (D. melanogaster)UAS-miR-996Bloomington Drosophila Stock CenterBDSC:60653
Genetic reagent (D. melanogaster)UAS-miR-998Bloomington Drosophila Stock CenterBDSC:63043
Genetic reagent (D. melanogaster)UAS-miR-306-spongeBloomington Drosophila Stock CenterBDSC:61424
Genetic reagent (D. melanogaster)UAS-miR-79-spongeBloomington Drosophila Stock CenterBDSC:61387
Genetic reagent (D. melanogaster)UAS-Luciferase RNAiBloomington Drosophila Stock CenterBDSC:31603
Genetic reagent (D. melanogaster)UAS-aop RNAiBloomington Drosophila Stock CenterBDSC:34909
Genetic reagent (D. melanogaster)UAS-pde1c RNAiBloomington Drosophila Stock CenterBDSC:55925
Genetic reagent (D. melanogaster)UAS-atf3 RNAiBloomington Drosophila Stock CenterBDSC:26741
Genetic reagent (D. melanogaster)UAS-mei-P26 RNAiBloomington Drosophila Stock CenterBDSC:57268
Genetic reagent (D. melanogaster)UAS-chn RNAiBloomington Drosophila Stock CenterBDSC:26779
Genetic reagent (D. melanogaster)UAS-chinmo RNAiBloomington Drosophila Stock CenterBDSC:26777
Genetic reagent (D. melanogaster)UAS-RNF146 RNAiBloomington Drosophila Stock CenterBDSC:40882
Genetic reagent (D. melanogaster)UAS-bcd RNAiBloomington Drosophila Stock CenterBDSC:33886
Genetic reagent (D. melanogaster)UAS-CG1358 RNAiBloomington Drosophila Stock CenterBDSC:64848
Genetic reagent (D. melanogaster)UAS-miR-306FlyORFFlyORF: F002214
Genetic reagent (D. melanogaster)UAS-TnksCore Facility of Drosophila Resource and Technology, Center for Excellence in Molecular Cell Science, Chinese Academy of SciencesN/A
Cell line (D. melanogaster)S2ATCCCat #CRL-1963
AntibodyAnti-phospho-JNK (rabbit monoclonal)Cell Signaling TechnologyCat #46681:100
AntibodyAnti-β-galactosidase (chicken polyclonal)AbcamCat #ab93611:1000
AntibodyAnti-cleaved Drosophila Dcp-1 (Asp216) (rabbit polyclonal)Cell Signaling TechnologyCat #95781:100
AntibodyGoat anti-rabbit secondary antibody, Alexa Fluor 647Thermo Fisher ScientificCat #A327331:250
AntibodyGoat anti-chicken secondary antibody, Alexa Fluor 647Thermo Fisher ScientificCat #A214491:250
AntibodyAnti-α-tubulin (mouse monoclonal)Sigma-AldrichCat #T51681:5000
AntibodyAnti-phospho-JNK (rabbit polyclonal)Cell Signaling TechnologyCat #92511:1000
AntibodyAnti-JNK (mouse monoclonal)Santa Cruz BiotechnologyCat #sc-73451:1000
AntibodyAnti-RNF146 (rabbit polyclonal)GenScript antibody serviceN/ARaised in rabbits against peptide HSGGGSGEDPAVGSC,1:2000
AntibodyAnti-V5 tag (mouse monoclonal)Thermo Fisher ScientificCat #R960-251:5000
AntibodyAnti-myc tag (rabbit polyclonal)MBLCat #5621:1000
AntibodyHorse anti-mouse IgG, HRP-linked antibodyCell Signaling TechnologyCat #70761:5000
AntibodyGoat anti-rabbit IgG, HRP-linked antibodyCell Signaling TechnologyCat #70741:5000
Commercial assay or kitDAPI-containing SlowFade Gold Antifade ReagentThermo Fisher ScientificCat #S36937
Commercial assay or kitFuGene HD transfection reagentPromegaCat #PRE2311
OtherCHXSanta Cruz BiotechnologyCat #SC-350850 μg/ml

Data availability

All relevant data are within the paper and its Supporting Information files. All the numerical data that are represented as a graph in a figure are provided in the Source Data file.

References

    1. Croce CM
    (2008) Oncogenes and cancer
    The New England Journal of Medicine 358:502–511.
    https://doi.org/10.1056/NEJMra072367

Decision letter

  1. Erika A Bach
    Reviewing Editor; New York University School of Medicine, United States
  2. Utpal Banerjee
    Senior Editor; University of California, Los Angeles, United States
  3. Erika A Bach
    Reviewer; New York University School of Medicine, United States

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Tumor elimination by clustered microRNAs miR-306 and miR-79 via non-canonical activation of JNK signaling" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Erika A Bach as Reviewing Editor and (Reviewer #1), and the evaluation has been overseen by Utpal Banerjee as the Senior Editor.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1. Show whether 1x or 2x copies of miR-306 or miR-79 cause death in larval or pupal eye discs.

2. Assess whether Rnf146 protein increases in miR 9c/306/79/9b loss-of-function clones in the eye disc.

3. Ensure that the same number of UAS transgenes are present in genotypes that are being compared; this might result in repeating experiments.

4. Improve Western blots of pJNK by normalizing to the level of total JNK (not α-tubulin) in the sample and increasing sample size to at least three independent replicates. The Western blot data should be displayed as a graph of normalized p-JNK levels with error bars and statistics.

5. Improve the Rnf146 Western blot in Figure 5F. At a bare minimum, there should be at least three replicates and quantification in a graph.

6. Assess whether these miRNAs enhance normally-occurring JNK activity, for example during dorsal closure in the embryo.

7. Many controls are not shown. These include but are not limited to:

– Figure 1 needs wild-type control (Luc) clones and quantification.

– Figure 2 needs wild-type control (Luc) clones and quantification (to the block of panels in Figure 2A-E) and p35-expressing clones and quantification (to the block of panels in Figure 2. F-I).

– Figure 3 needs wild-type control clones (Luc alone), and p35-expressing clones the first block of panels (A-I). In the Q-X section, the authors should add Luc alone clones, bsk-DN clones, Ras/dlg clones with quantification. M-P should show control eyes (GMR/+).

– Figure 4 A-E and Figure 4-Figure Sup 1, wild-type control clones are required.

– Figure 5, Ras-Dlg and dRNF146-OE clones should be included.

– Figure 6C, D, should include Tnks alone expressing clones.

8. Show expression of JNK targets – TRE-reporters, pJNK, or puc-lacZ – in miR-306 and 79 clones and in PVR-act, Src64B, Hel25E, and Mahj clones.

9. Show that miR-306 and 79 enhance JNK activity in Ras-Dlg tumors by showing the expression of JNK targets as the TRE-reporters, pJNK, or puc-lacZ.

10. Show that Ras-Dlg+miR-306/79+dRNF146 clones are the same size as the Ras-Dlg clones. Currently, this does not seem to be the case (compare Fig5G with Figure 5K or M).

11. Show that JNK activity levels are upregulated when dRNF146 is downregulated in those tumors.

12. Show that Tnks overexpression alone does not affect normal growth in otherwise wild-type clones.

13. Show that miR-309/79 modulation affects Tnks protein levels.

Reviewer #1 (Recommendations for the authors):

In the current study, Wang and colleagues perform a microRNA (miR) screen to find miRs that can suppress tumors caused by RasV12 and loss of polarity genes (i.e., discs large (dlg) or lethal giant larva (lgl)). This screen identified 12 miRs that when overexpressed suppressed the growth of RasV12 dlg-/- (or lgl-/-) tumors in the eye disc. They went on to characterize a cluster called miR 9c/306/79/9b. Clonal overexpression of miR-306 or miR-79 suppressed the tumors but did not greatly suppress growth of wild-type tissue. The authors went on to show that clonal overexpression of miR-306 or of miR-79 caused cell death in the tumors but did not cause death in WT tissue. Further experiments revealed that these miRs interact with cell polarity and not with RasV12. The authors then show that these miRs cause up regulation of JNK signaling as inhibiting JNK signaling abrogated the tumor-suppressing ability of these miRs. These miRs also enhanced JNK signaling in several other kinds of tumors that depend on JNK. They use an algorithm targetscanfly to search for miR binding sites and found 11 mRNAs that were predicted to be targets of both miR-306 and miR-79. They were able to test nine of these genes through RNA interference and only one of them Rnf146 caused JNK activation when depleted. They used luciferase assays in cultured cells to show that there were two binding sites in the 3'UTR of Rnf146 for these miRs. Knockdown of Rnf146 did not block the growth of normal tissue but it did significantly suppress the growth of RasV12 dlg-/- mutant cells. Rnf146 is a ubiquitin ligase that interacts with tankyrases (TNKs) – poly-ADP-ribose polymerases – to target proteins for degradation, and the authors next examined the role of TNKs in their tumor model. They used biochemistry to show that TNKs activate JNK by phosphorylation in S2 cells and that RNF146 downregulates TNK protein as well as JNK activation. Overexpression of TNKs in tumors suppresses their growth. Finally, they used biochemistry to show that when they blocked protein synthesis and depleted Rnf146, TNK protein levels remained relatively stable. These data support their model that miR-306 and -79 directly target Rnf146, which results in elevated TNK and this induces JNK signaling to cause cell death.

The novelty of this study is finding the connection between miR-306 and miR-79 and Rnf146. By contrast, it was already known that TNKs activates JNK signaling (Feng et al. 2018; Li et al. 2018) and that Rnf146 degrades TNKs (Gultekin, Y., Steller, H., 2019, Figure S2E-F, PMID: 30796047). The lattermost result is not acknowledged by the authors. Gultekin and Steller mis-expressed TNKs in eye discs in a WT or an RNF146 mutant background (Iduna-/- which is adult viable) and then performed Western blots on eye discs. They found that TNK protein levels were significantly higher in the Rnf146 mutant background than in WT.

There are several issues that I think should be addressed.

Issues with experiments/text:

1. The authors should acknowledge the results of Gultekin and Steller in the manuscript and compare results.

2. The authors state that over-expression of miR-306 or miR-79 (presumably using 1 copy of the transgene) does not reduce growth or WT cells. However, the results in Figure 5, figure supplement 3G-J does not support this model. In these experiments, the authors over-expressed 2 copies of UAS-miR and adult eyes from GMR>2x miR-306 or GMR>2x miR-79 are noticeably smaller than WT and are rough. These results suggest that miR-306 and miR-79 do indeed affect WT cells, which then may make it less likely that miR over-expression can be used as cancer therapy. Can the authors examine death cells in GMR>2x miR-306 or GMR>2x miR-79 larval or pupal discs? Alternatively, what happens when only 1 copy of the UAS-miR is over-expressed?

3. The authors depleted Rnf146 from GMR>Eiger and saw an enhancement of the eye phenotype. I might be confused, but I thought that it would be better to over-express Rnf146 in GMR>Eiger and look for suppression of the small eye phenotype.

4. Most of the experiments are over-expression. Does miR-306 or miR-79 normally regulate levels of Rnf-146 protein? Can you make a clone of the miR 9c/306/79/9b and see Rnf146 protein levels increase?

5. The Rnf146 null allele is adult viable. It might be beyond the scope of this work, but if you made RasV12, dlg-/- clones in an Rnf146 null mutant, the tumors should significantly smaller than in a heterozygous background.

Issues with figures:

1. Figure 1, figure supplement 1, panel AI: the authors need to mention that the dashed horizontal line at 60% comes from the RasV12 dlg-/- clones in Figure 1F.

2. Figure 2: Please show Dcp-1 in red or magenta in panels A'-D' as it is very difficult to see white on green.

3. Figure 2: panel I – there should be the same number of UAS transgenes in all the genotypes.

4. 4. Figure 2, figure supplement 1: Please show Dcp-1 in red or magenta in panels A'-D' as it is very difficult to see white on green.

5. Figure 3: panels T and X, there should be the same number of UAS transgenes in all the genotypes.

6. Figure 3, panel J: The authors should show a close up of pJNK in WT wing discs with Luc clones as panel J".

7. Figure 3, figure supplement 2, panel A: The authors should show a close up of pJNK in WT wing discs with Luc clones as panel A".

8. Figure 3, figure supplement 2, panel D: There are several issues with this figure. (1) pJNK levels should be normalize to the level of total JNK in the sample. It is possible that the lower level of pJNK is a result of less JNK protein in GMR>Luc compared to JNK in GMR>miR-306 or GMR-miR-79 and knowing the total level of JNK protein in these cells would allow them to disprove this possibility. Currently, the authors normalize with α-tubulin. (2) The Western blot data should be displayed as a graph of normalized p-JNK levels with error bars and statistics.

9. Figure 5, panels B' and C': Please show puc-lacZ in red or magenta in panels A'-D' as it is very difficult to see white on green.

10. Figure 5, panel F: The Western blot data should be displayed as a graph of normalized dRNF146 levels with error bars and statistics.

11. Figure 5, figure supplement 1, panels A'-F': Please show puc-lacZ in red or magenta as it is very difficult to see white on green.

12. The source files are in a.gel format, which I cannot access. Would you please upload them as.tif or.jpeg?

13. Include a file with complete genotypes for all figures.

Reviewer #2 (Recommendations for the authors):

This is a very complete manuscript. It requires only very few revisions and is appropriate for eLife.

1. The authors showed that overexpression of miR-79 and -306 alone was not sufficient to induce a significant phenotype on eye morphology. However, JNK activity is usually not active in eye discs and therefore it cannot be further enhanced. Can expression of these miRNAs enhance normally occurring JNK activity during normal development? One example where the author can address this question is dorsal closure during embryogenesis.

2. The authors mention that Tankyrase promotes K63-polyubiquitylation of JNK. Is it known how Tankyrase mediates this effect? Even if not, it is worth mentioning this in the text.

3. The authors did an excellent job in quantifying every experiment shown. However, in the Method section, they did not explain how the expression levels (Dcp-1, pJNK, puc-lacZ, etc.) were measured and quantified.

Reviewer #3 (Recommendations for the authors):

The experiments presented lack some controls. This will allow a more complete comparison between the genetic conditions analyzed.

– Figure 1 should show, as panel A, wild type control clones (Luc alone). The quantification of those clones should be shown in panel F.

– Figure 2: wild type control clones should be added in the 1st block of results (A-E). p35-expressing clones should be used as controls in the second group of results (F-I).

– Figure 3: the first block of results (A-I) should also include wild type control clones (Luc alone), and p35-expressing clones. In the Q-X section, Luc alone clones, bsk-DN clones, Ras/dlg clones should be shown and quantified. M-P should show control eyes (GMR/+).

– Figure 4 A-E, and Figure 4-Figure Sup 1, wild type control clones are required.

– Figure 5, Ras-Dlg and dRNF146-OE clones should be included.

– Figure 6C, D, hould include Tnks alone expressing clones.

To conclude that "miR-306 and miR-79 suppress tumor growth by enhancing JNK signaling", the authors should show that miR-306 and 79 enhance JNK activity in tumors by showing the expression of JNK targets as the TRE-reporters, pJNK, or puc-lacZ.

The title of section "miR-306 and miR-79 enhance JNK signaling in different types of tumors" is misleading and confusing. Among the 4 conditions analyzed – namely, PVR-act, Src64B, Hel25E, and Mahj, only the PVR-act condition (although it lacks the proper control) seems to cause oncogenic growth. As previously mentioned, to claim that those miRNAs enhance JNK activity, the authors should show the expression of JNK targets as the TRE-reporters, pJNK, or puc-lacZ.

Figure 4 K-O. The authors show that "non-autonomous overgrowth of surrounding wild-type tissue by Src64B-overexpressing clones". Is this observation relevant for this manuscript? It seems to come out of the blue and I can´t find the connection with the rest of results presented here.

Related to the regulation of dRNF146 by miRNA-305/79, the results shown in D and E show that the predicted binding site is sensitive to the levels of those miRNAs. However, results showing that the proposed regulation is indeed taking place in vivo and is physiologically relevant are missing. The western presented in Figure 5F is very poor. Results with better quality need to be provided to show that convincingly. In line with that, showing the protein levels in miR-306 and 79-expressing clones in the eye disc will provide a convincing piece of data.

The authors claim: "Furthermore, overexpression of dRNF146 cancelled the tumor-suppressive effect of miR-306 or miR-79 on RasV12/dlg-/- tumors (Figure 5J-M, quantified in Figure 5N)." To show that convincingly, the authors need to show that Ras-Dlg+miR-306/79+dRNF146 clones present the same size as the Ras-Dlg clones. At glance, this does not seem to be the case (compare Fig5G with Figure 5K or M).

After that, the authors state: "Together, these data indicate that miR-306 and miR-79 directly target dRNF146 mRNA, thereby enhancing JNK signaling activity and thus exerting the tumor-suppressive effects." The authors make different correlations to reach that conclusion but do not show evidence proving that dRNF146 depletion is in fact enhancing JNK activity in those tumors. To conclude that, the authors should prove that JNK activity levels are upregulated when dRNF146 is downregulated in those tumors.

To demonstrate their model, the authors should show that Tnks overexpression alone does not affect normal growth in otherwise wild type clones. The authors should also show that miR-309/79 modulation affect Tnks protein levels.

https://doi.org/10.7554/eLife.77340.sa1

Author response

Essential revisions:

1. Show whether 1x or 2x copies of miR-306 or miR-79 cause death in larval or pupal eye discs.

We agree with the comment. Following the suggestion, we have now tested whether 1x or 2x copies of miR-306 or miR-79 cause death in larval eye discs. Our data indicate that, although overexpression of one copy of miR-306 or miR-79 in the eyes had no significant effect on the number of dying cells, overexpression of 2 copies of miR-306 or miR-79 in the eyes significantly increased the number of dying cells. We have now included these new data in Figure 5—figure supplement 4G-M (quantified in Figure 5—figure supplement 4N).

2. Assess whether Rnf146 protein increases in miR 9c/306/79/9b loss-of-function clones in the eye disc.

We do agree with the comment that we should test whether Rnf146 protein increases when miR-306/79 is suppressed. Unfortunately, neither the previous RNF146 antibody (a gift from Hermann Steller) nor a new RNF146 antibody (raised in rabbits against the peptide HSGGGSGEDPAVGSC) worked well in the immunofluorescence assay. Therefore, as an alternative method, we performed Western blots to detect the Rnf146 protein level using the new RNF146 antibody. As shown in Figure 5—figure supplement 3 in the revised manuscript, suppression of miR-306 and miR-79 function by using miRNA sponges indeed increased the endogenous levels of RNF146 protein in the adult eyes.

3. Ensure that the same number of UAS transgenes are present in genotypes that are being compared; this might result in repeating experiments.

We agree with the comment. Following the suggestion, we have now performed a series of new control experiments to make the same number of UAS transgenes present in genotypes that are being compared (please see Figures 1-6) unless it is technically too difficult by Drosophila genetics in some experiments (e.g., the experiments in Figure 3—figure supplement 3).

4. Improve Western blots of pJNK by normalizing to the level of total JNK (not α-tubulin) in the sample and increasing sample size to at least three independent replicates. The Western blot data should be displayed as a graph of normalized p-JNK levels with error bars and statistics.

We agree with the comment. Following the suggestion, we have now repeated all the Western blot experiments that used the pJNK antibody and were normalized by the total JNK level. All the Western blot data have now been repeated for three times and displayed as a graph with error bars and statistics in the revised Figure 6, Figure 3—figure supplement 2 and Figure 6—figure supplement 1.

5. Improve the Rnf146 Western blot in Figure 5F. At a bare minimum, there should be at least three replicates and quantification in a graph.

We agree with the comment. Following the suggestion, we have now used a new RNF146 antibody (raised in rabbits against the peptide HSGGGSGEDPAVGSC, GenScript antibody service, Nanjing, China). As shown in the revised Figure 5F, we believe that the data quality has now been significantly improved. We repeated this experiment for three times and quantified the data as shown in the revised Figure 5G.

6. Assess whether these miRNAs enhance normally-occurring JNK activity, for example during dorsal closure in the embryo.

We appreciate the valuable comment. Following the suggestion, we have now examined whether expression of these miRNAs enhance normally occurring JNK activity during normal development. The pnr-GAL4 fly strain specifically expresses GAL4 in the wing discs in a broad domain corresponding to the central presumptive notum during metamorphosis. Knocking down Hep, the Drosophila JNK kinase, using the pnr-GAL4 driver generates a split-thorax phenotype caused by reduced JNK signaling. On the contrary, overexpression of Hep or Eiger, the Drosophila TNF that activates JNK signaling, using pnr-GAL4 generates a small-scutellum phenotype caused by elevated JNK signaling. Similar to the Hep or Eiger-induced phanotype, ectopic expression of miR-306 or 79 by the pnr-GAL4 driver resulted in a small-scutellum phenotype, indicating that miR-306/79 enhances normally-occurring JNK activity. We have now included these new data as Figure 4—figure supplement 3A-C (quantified in Figure 4—figure supplement 3D) in the revised manuscript.

7. Many controls are not shown. These include but are not limited to:

– Figure 1 needs wild-type control (Luc) clones and quantification.

– Figure 2 needs wild-type control (Luc) clones and quantification (to the block of panels in Figure 2A-E) and p35-expressing clones and quantification (to the block of panels in Figure 2. F-I).

– Figure 3 needs wild-type control clones (Luc alone), and p35-expressing clones the first block of panels (A-I). In the Q-X section, the authors should add Luc alone clones, bsk-DN clones, Ras/dlg clones with quantification. M-P should show control eyes (GMR/+).

– Figure 4 A-E and Figure 4-Figure Sup 1, wild-type control clones are required.

– Figure 5, Ras-Dlg and dRNF146-OE clones should be included.

– Figure 6C, D, should include Tnks alone expressing clones.

We agree with the comment. Following the suggestion, we have now added new controls in the revised Figures (please see Figures 1-6).

8. Show expression of JNK targets – TRE-reporters, pJNK, or puc-lacZ – in miR-306 and 79 clones and in PVR-act, Src64B, Hel25E, and Mahj clones.

We agree with the comment. Following the suggestion, we have now tested whether miR-306 and 79 enhance JNK activity in all these tumors or cell competition models using the pJNK antibody. As shown in the revised Figure 4—figure supplement 2D-P, miR-306 or 79 indeed enhanced JNK activity in all these tumors or cell competition models.

9. Show that miR-306 and 79 enhance JNK activity in Ras-Dlg tumors by showing the expression of JNK targets as the TRE-reporters, pJNK, or puc-lacZ.

We agree with the comment. Following the suggestion, we have now tested whether miR-306 and 79 enhance JNK activity in Ras-Dlg tumors using the pJNK antibody. As shown in the revised Figure 4—figure supplement 2A-C (quantified in 2P), both miR-306 and 79 enhanced JNK activity in Ras-Dlg tumors.

10. Show that Ras-Dlg+miR-306/79+dRNF146 clones are the same size as the Ras-Dlg clones. Currently, this does not seem to be the case (compare Fig5G with Figure 5K or M).

We sincerely thank the reviewers and editors for the comment. We indeed noticed that Ras-Dlg+miR-306/79+dRNF146 clones are smaller than Ras-Dlg clones. We have now added a discussion for the possible reason for this phenomenon in the second paragraph of the “Discussion” section in the revised manuscript. Our study identified several putative co-target genes of miR-306 and miR-79 (Figure 5A). Interestingly, some of these genes (Atf3, chinmo, and chn) have been reported to be involved in tumor growth in Drosophila. Atf3 encodes an AP-1 transcription factor, which was shown to be a polarity-loss responsive gene acting downstream of the membrane-associated Scrib polarity complex (Donohoe et al., 2018). Knockdown of Atf3 suppresses growth and invasion of RasV12/scrib-/- tumors in the eye-antennal discs (Atkins et al., 2016). Chinmo is a BTB-zinc finger oncogene that is up-regulated by JNK signaling in tumors (Doggett et al., 2015). Although loss of chinmo does not significantly suppress tumor growth, overexpression of chinmo with RasV12 or activated Notch is sufficient to promote tumor growth in the eye-antennal discs (Doggett et al., 2015). Chn encodes a zinc finger transcription factor that cooperates with scrib-/- to promote tumor growth (Turkel et al., 2013). Although we found that knockdown of these genes did not activate JNK signaling, it is possible that these putative target genes also contribute to the miR-306/miR-79-induced tumor suppression and therefore Ras-Dlg+miR-306/79+dRNF146 clones are smaller than Ras-Dlg clones.

11. Show that JNK activity levels are upregulated when dRNF146 is downregulated in those tumors.

We agree with the comment. Following the suggestion, we have now tested whether JNK activity levels are upregulated when dRNF146 is downregulated in Ras-Dlg tumors using the pJNK antibody. As shown in the revised Figure 5—figure supplement 5, knocking down RNF146 indeed enhanced JNK activity in Ras-Dlg tumors.

12. Show that Tnks overexpression alone does not affect normal growth in otherwise wild-type clones.

We sincerely thank the reviewers and editors for the comment. Following the suggestion, we have now tested whether Tnks overexpression alone affect normal growth. As shown in Figure 6G-H, Tnks overexpression alone resulted in smaller clone size compared to control. However, considering that overexpression of Tnks alone shows larger clone size than RasV12/dlg-/-+Tnks (Figure 6H and J, quantified in Figure 6K), we believe that Tnks suppresses growth of RasV12/dlg-/- tumors by cooperating with JNK signaling.

13. Show that miR-309/79 modulation affects Tnks protein levels.

We agree with the comment. Following the suggestion, we have now analyzed the Tnks protein levels when miR-306 or 79 is overexpressed. As shown in Figure 6—figure supplement 1, overexpression of miR-306 or 79 indeed significantly upregulated the Tnks protein level.

Reviewer #1 (Recommendations for the authors):

There are several issues that I think should be addressed.

Issues with experiments/text:

1. The authors should acknowledge the results of Gultekin and Steller in the manuscript and compare results.

We agree with the comment. Actually, we had already described the results of Gultekin and Steller in the first paragraph of the “RNF146 promotes Tnks degradation” part of the “Results” section. However, following the suggestion, we have now added a sentence “A pervious study has indicated that Tnks protein levels were significantly higher in the Rnf146 mutant background than in wild-type (Gultekin and Steller, 2019).” in the second paragraph of the same part.

The main difference between their and our studies is that their study did not clarify whether the up-regulation of Tnks is caused by either elevated Tnks protein synthesis or reduced Tnks protein degradation. In our study, using the cycloheximide (CHX) assay, we clarified that RNF146 promotes the degradation of Tnks protein.

2. The authors state that over-expression of miR-306 or miR-79 (presumably using 1 copy of the transgene) does not reduce growth or WT cells. However, the results in Figure 5, figure supplement 3G-J does not support this model. In these experiments, the authors over-expressed 2 copies of UAS-miR and adult eyes from GMR>2x miR-306 or GMR>2x miR-79 are noticeably smaller than WT and are rough. These results suggest that miR-306 and miR-79 do indeed affect WT cells, which then may make it less likely that miR over-expression can be used as cancer therapy. Can the authors examine death cells in GMR>2x miR-306 or GMR>2x miR-79 larval or pupal discs? Alternatively, what happens when only 1 copy of the UAS-miR is over-expressed?

We agree with the comment. As responded in the Essential Revisions #1, although overexpression of one copy of miR-306 or miR-79 in the eyes had no significant effect on the number of dying cells, overexpression of 2×miR-306 or 2×miR-79 significantly increased the number of dying cells (Figure 5—figure supplement 4G-M, quantified in Figure 5—figure supplement 4N).

3. The authors depleted Rnf146 from GMR>Eiger and saw an enhancement of the eye phenotype. I might be confused, but I thought that it would be better to over-express Rnf146 in GMR>Eiger and look for suppression of the small eye phenotype.

We thank the reviewer for the comment. Our aim was to prove that depletion of Rnf146 have similar effect on the eye phenotype with overexpression of miR-306 or miR-79. Since Rnf146 is a component of the non-canonical JNK pathway, a lateral branch but not the major JNKKK-JNKK-JNK pathway, overexpression of Rnf146 may not be strong enough to suppress GMR>Eiger induced small-eye phenotype.

4. Most of the experiments are over-expression. Does miR-306 or miR-79 normally regulate levels of Rnf-146 protein? Can you make a clone of the miR 9c/306/79/9b and see Rnf146 protein levels increase?

We agree with the comment. As responded in the Essential Revisions #2, neither the previous RNF146 antibody (a gift from Hermann Steller) nor the new RNF146 antibody (raised in rabbits against the peptide HSGGGSGEDPAVGSC) worked well in the immunofluorescence assay. As an alternative method, we performed Western blots to detect the Rnf146 protein levels using the new RNF146 antibody. As shown in Figure 5—figure supplement 3, suppression of miR-306 and miR-79 function using miRNA sponges indeed promoted the endogenous levels of RNF146 protein in adult eyes.

5. The Rnf146 null allele is adult viable. It might be beyond the scope of this work, but if you made RasV12, dlg-/- clones in an Rnf146 null mutant, the tumors should significantly smaller than in a heterozygous background.

We sincerely thank the reviewer for the comment. However, it turned out that it is so hard to generate RasV12+dlg-/- clones in Rnf146-/- larvae. We have tried to establish flies for several times, but we were not able to succeed in making dlgm52, FRT19A; UAS-RasV12; Rnf146-/TM6B or TM3 flies.

Issues with figures:

1. Figure 1, figure supplement 1, panel AI: the authors need to mention that the dashed horizontal line at 60% comes from the RasV12 dlg-/- clones in Figure 1F.

2. Figure 2: Please show Dcp-1 in red or magenta in panels A'-D' as it is very difficult to see white on green.

3. Figure 2: panel I – there should be the same number of UAS transgenes in all the genotypes.

4. 4. Figure 2, figure supplement 1: Please show Dcp-1 in red or magenta in panels A'-D' as it is very difficult to see white on green.

5. Figure 3: panels T and X, there should be the same number of UAS transgenes in all the genotypes.

6. Figure 3, panel J: The authors should show a close up of pJNK in WT wing discs with Luc clones as panel J".

7. Figure 3, figure supplement 2, panel A: The authors should show a close up of pJNK in WT wing discs with Luc clones as panel A".

8. Figure 3, figure supplement 2, panel D: There are several issues with this figure. (1) pJNK levels should be normalize to the level of total JNK in the sample. It is possible that the lower level of pJNK is a result of less JNK protein in GMR>Luc compared to JNK in GMR>miR-306 or GMR-miR-79 and knowing the total level of JNK protein in these cells would allow them to disprove this possibility. Currently, the authors normalize with α-tubulin. (2) The Western blot data should be displayed as a graph of normalized p-JNK levels with error bars and statistics.

9. Figure 5, panels B' and C': Please show puc-lacZ in red or magenta in panels A'-D' as it is very difficult to see white on green.

10. Figure 5, panel F: The Western blot data should be displayed as a graph of normalized dRNF146 levels with error bars and statistics.

11. Figure 5, figure supplement 1, panels A'-F': Please show puc-lacZ in red or magenta as it is very difficult to see white on green.

12. The source files are in a.gel format, which I cannot access. Would you please upload them as.tif or.jpeg?

13. Include a file with complete genotypes for all figures.

We sincerely thank the reviewer for the comments. Following the suggestions, we have now added new data, revised the figures and descriptions, uploaded the source files for Western blots as.tif, and included files with complete genotypes for each figure as source files in the revised manuscript.

Reviewer #2 (Recommendations for the authors):

This is a very complete manuscript. It requires only very few revisions and is appropriate for eLife.

1. The authors showed that overexpression of miR-79 and -306 alone was not sufficient to induce a significant phenotype on eye morphology. However, JNK activity is usually not active in eye discs and therefore it cannot be further enhanced. Can expression of these miRNAs enhance normally occurring JNK activity during normal development? One example where the author can address this question is dorsal closure during embryogenesis.

We appreciate the valuable comment. As responded in the Essential Revisions #6, we have now examined whether expression of these miRNAs enhance normally occurring JNK activity during normal development. The pnr-GAL4 driver strain specifically expresses GAL4 in the wing discs in a broad domain corresponding to the central presumptive notum during metamorphosis. Knocking down Hep, the Drosophila JNK kinase, using the pnr-GAL4 driver generates a split-thorax phenotype caused by suppressing JNK signaling. On the contrary, ectopic expression of Hep or Eiger using pnr-GAL4 generates a small-scutellum phenotype caused by activation of JNK signaling. Similarly to Hep or Eiger overexpression, ectopic expression of miR-306 or 79 driven by pnr-GAL4 resulted in a small-scutellum phenotype (please see Figure 4—figure supplement 3A-C, quantified in Figure 4—figure supplement 3D in the revised manuscript). These data indicate that miR-306/79 enhances normally-occurring JNK activity.

2. The authors mention that Tankyrase promotes K63-polyubiquitylation of JNK. Is it known how Tankyrase mediates this effect? Even if not, it is worth mentioning this in the text.

We thank the reviewer for the comment. As reported by Ping Li et al., Tnks mediates Poly-ADP ribosylation of JNK, triggers K63-linked poly-ubiquitination of JNK and thereby enhances JNK kinase activity (Li et al., 2018). Following this suggestion, we have now mentioned this in the “RNF146 promotes Tnks degradation” part of the “Results” section in the revised manuscript.

3. The authors did an excellent job in quantifying every experiment shown. However, in the Method section, they did not explain how the expression levels (Dcp-1, pJNK, puc-lacZ, etc.) were measured and quantified.

We thank the reviewer for the comment. The cleaved Dcp-1-positive cell number and pJNK-positive area were calculated using ImageJ and Prism 8 (Graphpad). Following the suggestion, we have now mentioned this in the “Histology” part of the “Materials and Methods” section in the revised manuscript.

Reviewer #3 (Recommendations for the authors):

The experiments presented lack some controls. This will allow a more complete comparison between the genetic conditions analyzed.

– Figure 1 should show, as panel A, wild type control clones (Luc alone). The quantification of those clones should be shown in panel F.

– Figure 2: wild type control clones should be added in the 1st block of results (A-E). p35-expressing clones should be used as controls in the second group of results (F-I).

– Figure 3: the first block of results (A-I) should also include wild type control clones (Luc alone), and p35-expressing clones. In the Q-X section, Luc alone clones, bsk-DN clones, Ras/dlg clones should be shown and quantified. M-P should show control eyes (GMR/+).

– Figure 4 A-E, and Figure 4-Figure Sup 1, wild type control clones are required.

– Figure 5, Ras-Dlg and dRNF146-OE clones should be included.

– Figure 6C, D, hould include Tnks alone expressing clones.

We sincerely thank the reviewer for these comments. Following the suggestion, we have now added many controls and revised the figures in the revised manuscript.

To conclude that "miR-306 and miR-79 suppress tumor growth by enhancing JNK signaling", the authors should show that miR-306 and 79 enhance JNK activity in tumors by showing the expression of JNK targets as the TRE-reporters, pJNK, or puc-lacZ.

We agree with the comment. Following the suggestion, as responded in the Essential Revisions #9, we have now tested whether miR-306 and 79 enhance JNK activity in Ras-Dlg tumors using the pJNK antibody. As shown in the revised Figure 4—figure supplement 2A-C, both miR-306 and 79 indeed enhanced JNK activity in Ras-Dlg tumors.

The title of section "miR-306 and miR-79 enhance JNK signaling in different types of tumors" is misleading and confusing. Among the 4 conditions analyzed – namely, PVR-act, Src64B, Hel25E, and Mahj, only the PVR-act condition (although it lacks the proper control) seems to cause oncogenic growth. As previously mentioned, to claim that those miRNAs enhance JNK activity, the authors should show the expression of JNK targets as the TRE-reporters, pJNK, or puc-lacZ.

We appreciate the valuable comment. We have now revised the title to “miR-306 and miR-79 enhance JNK signaling stimulated by different upstream signaling”. Following the suggestion, as responded in the Essential Revisions #8, we have now tested whether miR-306 and 79 enhance JNK activity in all these tumors or cell competition models using the pJNK antibody. As shown in the revised Figure 4—figure supplement 2D-O, miR-306 or 79 indeed enhanced JNK activity in all these tumors or cell competition models.

Figure 4 K-O. The authors show that "non-autonomous overgrowth of surrounding wild-type tissue by Src64B-overexpressing clones". Is this observation relevant for this manuscript? It seems to come out of the blue and I can´t find the connection with the rest of results presented here.

The non-autonomous overgrowth of surrounding wild-type tissue is caused by Src64B-overexpressing cells (Enomoto and Igaki, 2013). Coexpression of miR-9c/306/79/9b cluster, miR-306, or miR-79 kills Src64B-overexpressing cells in the eye-antennal discs (Figure 4H-L) and thus suppresses the non-autonomous overgrowth of surrounding wild-type tissue (Figure 4M-Q). This data indicated that miR-306 or miR-79 not only suppresses tumor growth caused by autonomous overgrowth (e.g., RasV12/dlg-/-, RasV12/lgl-/- and PVRact), but also suppresses tumor growth caused by non-autonomous overgrowth.

Related to the regulation of dRNF146 by miRNA-305/79, the results shown in D and E show that the predicted binding site is sensitive to the levels of those miRNAs. However, results showing that the proposed regulation is indeed taking place in vivo and is physiologically relevant are missing. The western presented in Figure 5F is very poor. Results with better quality need to be provided to show that convincingly. In line with that, showing the protein levels in miR-306 and 79-expressing clones in the eye disc will provide a convincing piece of data.

We agree with the comment. As responded in the Essential Revisions #2 and #5, we have now used a new RNF146 antibody (raised in rabbits against the peptide HSGGGSGEDPAVGSC, GenScript antibody service, Nanjing, China). As shown in the revised Figure 5F, we believe the data quality have now significantly improved. We repeated this experiment for three times and quantified the data as shown in the revised Figure 5G. Unfortunately, neither the previous RNF146 antibody (a gift from Hermann Steller) nor the new RNF146 antibody worked well in the immunofluorescence assay. As an alternative method, we performed Western blots to detect the Rnf146 protein levels using the new RNF146 antibody. As shown in Figure 5—figure supplement 3 in the revised manuscript, suppression of miR-306 and miR-79 functions using miRNA sponges indeed promoted the endogenous levels of RNF146 protein in the adult eyes.

The authors claim: "Furthermore, overexpression of dRNF146 cancelled the tumor-suppressive effect of miR-306 or miR-79 on RasV12/dlg-/- tumors (Figure 5J-M, quantified in Figure 5N)." To show that convincingly, the authors need to show that Ras-Dlg+miR-306/79+dRNF146 clones present the same size as the Ras-Dlg clones. At glance, this does not seem to be the case (compare Fig5G with Figure 5K or M).

We thank the reviewer for the comment. We have now replaced the word “cancelled” with “weakened” in the revised manuscript. As responded in the Essential Revisions #10, we indeed noticed that Ras-Dlg+miR-306/79+dRNF146 clones are smaller than Ras-Dlg clones. We have now added a discussion for the possible reason for this phenomenon in the second paragraph of the “Discussion” section in the revised manuscript. Our study identified several putative co-target genes of miR-306 and miR-79 (Figure 5A). Interestingly, some of these genes (Atf3, chinmo, and chn) have been reported to be involved in tumor growth in Drosophila. Atf3 encodes an AP-1 transcription factor which was shown to be a polarity-loss responsive gene acting downstream of the membrane-associated Scrib polarity complex (Donohoe et al., 2018). Knockdown of Atf3 suppresses growth and invasion of RasV12/scrib-/- tumors in the eye-antennal discs (Atkins et al., 2016). Chinmo is a BTB-zinc finger oncogene that is up-regulated by JNK signaling in tumors (Doggett et al., 2015). Although loss of chinmo does not significantly suppress tumor growth, overexpression of chinmo with RasV12 or activated Notch is sufficient to promote tumor growth in eye-antennal discs (Doggett et al., 2015). Chn encodes a zinc finger transcription factor that cooperates with scrib-/- to promote tumor growth (Turkel et al., 2013). Although we found that knockdown of these genes did not activate JNK signaling, it is possible that these putative target genes also contribute to the miR-306/miR-79-induced tumor suppression and thus Ras-Dlg+miR-306/79+dRNF146 clones are smaller than Ras-Dlg clones..

After that, the authors state: "Together, these data indicate that miR-306 and miR-79 directly target dRNF146 mRNA, thereby enhancing JNK signaling activity and thus exerting the tumor-suppressive effects." The authors make different correlations to reach that conclusion but do not show evidence proving that dRNF146 depletion is in fact enhancing JNK activity in those tumors. To conclude that, the authors should prove that JNK activity levels are upregulated when dRNF146 is downregulated in those tumors.

We agree with the comment. As responded in the Essential Revisions #11, we have now tested whether JNK activity levels are upregulated when dRNF146 is downregulated in Ras-Dlg tumors using the pJNK antibody. As shown in the revised Figure 5—figure supplement 5, knocking down RNF146 indeed enhanced JNK activity in Ras-Dlg tumors.

To demonstrate their model, the authors should show that Tnks overexpression alone does not affect normal growth in otherwise wild type clones. The authors should also show that miR-309/79 modulation affect Tnks protein levels.

We sincerely thank the reviewer for the comment. As responded in the Essential Revisions #12 and #13, we have now tested whether Tnks overexpression alone affect normal growth. As shown in Figure 6G-H, Tnks overexpression alone resulted in smaller clone size compared to control. However, considering that overexpression of Tnks alone shows larger clone size than RasV12/dlg-/-+Tnks (Figure 6H and J, quantified in Figure 6K), we believe that Tnks suppresses growth of RasV12/dlg-/- tumors by cooperating with JNK signaling. We also detected the Tnks protein levels when miR-306 or 79 is overexpressed. As shown in Figure 6—figure supplement 1, overexpression of miR-306 or 79 significantly upregulated Tnks protein level.

https://doi.org/10.7554/eLife.77340.sa2

Article and author information

Author details

  1. Zhaowei Wang

    1. State Key Laboratory of Biocontrol, School of Ecology, Sun Yat-sen University, Shenzhen, China
    2. Laboratory of Genetics, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Kyoto, Japan
    Contribution
    Conceptualization, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4491-8502
  2. Xiaoling Xia

    1. Laboratory of Genetics, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Kyoto, Japan
    2. Guangzhou Key Laboratory of Insect Development Regulation and Application Research, Institute of Insect Science and Technology & School of Life Sciences, South China Normal University, Guangzhou, China
    Contribution
    Conceptualization, Formal analysis, Investigation, Visualization, Writing - original draft
    Competing interests
    No competing interests declared
  3. Jiaqi Li

    Laboratory of Genetics, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Kyoto, Japan
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Tatsushi Igaki

    Laboratory of Genetics, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Kyoto, Japan
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    igaki.tatsushi.4s@kyoto-u.ac.jp
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5839-9526

Funding

MEXT/JSPS KAKENHI (20H05320)

  • Tatsushi Igaki

MEXT/JSPS KAKENHI (21H05284)

  • Tatsushi Igaki

MEXT/JSPS KAKENHI (21H05039)

  • Tatsushi Igaki

Japan Agency for Medical Research and Development (Project for Elucidating and Controlling Mechanisms of Aging and Longevity)

  • Tatsushi Igaki

Japan Agency for Medical Research and Development (20gm5010001)

  • Tatsushi Igaki

Takeda Science Foundation

  • Tatsushi Igaki

Fundamental Research Funds for the Central Universities (Sun Yat-sen University)

  • Zhaowei Wang

Fundamental Research Funds for the Central Universities (22hytd05)

  • Zhaowei Wang

Naito Foundation

  • Tatsushi Igaki

Japan Society for the Promotion of Science (Postdoctoral Fellowships for Research in Japan)

  • Zhaowei Wang

China Scholarship Council (for Research in Japan)

  • Xiaoling Xia

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank M Matsuoka and K Gomi for technical support, Hermann Steller, the Bloomington Drosophila Stock Center, the National Institute of Genetics Stock Center (NIG-FLY), the Drosophila Genomics and Genetic Resources (DGGR, Kyoto Institute of Technology), the Vienna Drosophila Resource Center (VDRC), and the Core Facility of Drosophila Resource and Technology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences for fly stocks and reagents. We also thank the members of the Igaki laboratory for discussions. This work was supported by grants from the MEXT/JSPS KAKENHI (grant numbers 20H05320, 21H05284, and 21H05039) to TI, Japan Agency for Medical Research and Development (Project for Elucidating and Controlling Mechanisms of Aging and Longevity; grant number 20gm5010001) to TI, the Takeda Science Foundation to TI, the Fundamental Research Funds for the Central Universities, Sun Yat-sen University to ZW (grant number 22hytd05), and the Naito Foundation to TI. ZW was supported by JSPS Postdoctoral Fellowships for Research in Japan, and XX was supported by China Scholarship Council for Research in Japan.

Senior Editor

  1. Utpal Banerjee, University of California, Los Angeles, United States

Reviewing Editor

  1. Erika A Bach, New York University School of Medicine, United States

Reviewer

  1. Erika A Bach, New York University School of Medicine, United States

Publication history

  1. Received: January 25, 2022
  2. Preprint posted: February 11, 2022 (view preprint)
  3. Accepted: October 11, 2022
  4. Accepted Manuscript published: October 12, 2022 (version 1)
  5. Version of Record published: October 27, 2022 (version 2)

Copyright

© 2022, Wang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Zhaowei Wang
  2. Xiaoling Xia
  3. Jiaqi Li
  4. Tatsushi Igaki
(2022)
Tumor elimination by clustered microRNAs miR-306 and miR-79 via noncanonical activation of JNK signaling
eLife 11:e77340.
https://doi.org/10.7554/eLife.77340
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