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

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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 no significant 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 Drosophila ring finger protein 146 (dRNF146). We found that DRNF146 promotes degradation of tankyrase (Tnks), an ADP-ribose polymerase that promotes JNK activation in a non-canonical manner. Thus, downregulation of dRNF146 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 non-canonical JNK pathway and thus eliminate tumors, providing a new miRNA-based strategy against cancer.

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  1. Evaluation Summary:

    This paper is of interest for cancer biologists studying the role microRNAs in tumor growth. The work provides links between over-expression of microRNAs, downregulation of a ubiquitin ligase, inhibition of JNK and reduced tumor growth. Some of the data are properly controlled and analyzed. However, the key claims of the manuscript are not entirely supported by the data, and additional controls are needed.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    Wang et al. investigated the roles of over-expressed microRNAs (miRs) in a Drosophila tumor model caused by concomitant clonal gain of RasV12 and clonal loss of Discs large in developing epithelia. They identified miR-306 and miR-79 from the same miR cluster as able to reduce tumor growth significantly when over-expressed in the tumor cells. These miRs were shown to target the 3'UTR of the ubiquitin ligase Rnf146, causing Rnf146 protein downregulation. It has already been established in the field that Rnf146 interacts with poly-ADP-ribose polymerases called Tankyrases (TNKs) to target proteins for degradation and that TNKs activate JNK signaling. The authors find that over-expressed Rnf146 can downregulate TNKs and JNK signaling in cultured S2 cells and in Drosophila tumors. The authors propose that miR-306 and -79 directly target Rnf146, which results in elevated TNK and this induces JNK signaling and causes the death of tumors.

    The novelty of this study is identifying Rnf146 as a target of miR-306 and miR-79. The connections between Rnf146-TNK and TNK-JNK were already known from the work of others. A main conclusion of the paper - that these miRs could be used as cancer therapeutics - is not strongly supported by the data because in some experiments over-expression of these miRs negatively affected WT cells, and this undermines the authors' conclusion that miR over-expression does not affect the growth of WT cells. Strengths of the paper are: the use of powerful clonal techniques to make, monitor and measure tumors, and the proper analysis and quantification of some of the results. However, some of the data analyses need to be clarified and extended.

  3. Reviewer #2 (Public Review):

    This is a very straightforward manuscript by the laboratory of Tatsushi Igaki in Kyoto, Japan. The authors identify a number of miRNAs whose expression in RasV12/dlg- tumors suppresses tumor growth. They also show that several of these miRNAs suppress RasV12/lgl- tumor growth. They focus on two of these miRNAs, miR-79 and miR-306, and worked out the mechanism by which these miRNAs act as tumor suppressors. First, they show that expression of these miRNAs induces apoptosis in RasV12/dlg- tumor and RasV12/lgl- clones. They then show that miR-79 and mi-306 increase JNK signaling in these tumor clones. Inhibition of JNK by bskDN restores tumor growth, suggesting that the tumor suppressive function of these miRNAs goes through JNK signaling. Interestingly also, the tumor suppressive function of these miRNA is dependent on the dlg- component and not on RasV12. They then show that other tumor types which are associated with increased JNK signaling (PVR, Src64B) can also be suppressed by expression of these miRNAs. Using bioinformatics, they identified 11 potential mRNA targets of these miRNAs. Only one of them, encoding the Ringfinger protein dRNF146, accounts for the increased JNK activation and the authors show that dRNF146 is a direct target of these miRNAs. It was previously shown that dRNF146 can target the poly-ADP-ribose polymerase Tankyrase for degradation. Tankyrase can promote JNK phosphorylation and activation in a non-canonical manner. The authors performed a number of elegant experiments and essentially confirmed that Tankyrase degradation is blocked in these tumor clones when miR-79 and -306 were overexpressed.

    Overall, this is a very nice and straightforward manuscript. It presents a tumor suppressor mechanism which exploits tumor-promoting JNK activity in tumor cells by overactivating it so that the tumor cells die by JNK-induced apoptosis. The mechanism in this manuscript is very well worked out. A large number of controls were done. The data are very convincing, very well quantified and well presented. The manuscript is also very well written and very easy to read. I did not detect any significant weakness.

  4. Reviewer #3 (Public Review):

    In this M, Wang and cols use the Drosophila Ras-Dlg tumor model to search for miRNAs with a tumor suppressive role. The authors identify two miRNAs - namely miR-306 and miR-79, as elements that reduce oncogenic growth but do not affect normal growth. After, the authors try to identify the mechanisms by which these miRNAs limit tumor growth. The authors claim that the axis dNRF146-Tnks enhances JNK activity inducing apoptosis in tumor cells. The authors propose an exciting synthetic lethality interaction between the tumor background and these miRNAs. However, the conclusions obtained are not well-supported by the results presented here.

    The authors claim that miR-306/79 over-expression and dRNF146 downregulation enhances JNK activation in tumor cells. This is based on different correlations but changes in JNK activity in those genetic contexts are not shown.

    The results suggesting that dRNF146 is a miR-306/79 target gene with physiological relevance are very poor.

    This work is based in the comparison between many very complex genetic backgrounds. However, most of those experiments lack the control experiments that will allow to compare those conditions properly.

    Finally, the connection between dRNF146, Tnks, and JNK has been previously established and it is not novel.