A MAPK/miR-29 Axis Suppresses Melanoma by Targeting MAFG and MYBL2

  1. Olga Vera
  2. Ilah Bok
  3. Neel Jasani
  4. Koji Nakamura
  5. Xiaonan Xu
  6. Nicol Mecozzi
  7. Ariana Angarita
  8. Kaizhen Wang
  9. Kenneth Y. Tsai
  10. Florian A. Karreth

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Abstract

The miR-29 family of microRNAs is encoded by two clusters, miR-29b1~a and miR-29b2~c, and is regulated by several oncogenic and tumor suppressive stimuli. While in vitro evidence suggests a tumor suppressor role for miR-29 in melanoma, the mechanisms underlying its deregulation and contribution to melanomagenesis have remained elusive. Using various in vitro systems, we show that oncogenic MAPK signaling paradoxically stimulates transcription of pri-miR-29b1~a and pri-miR-29b2~c, the latter in a p53-dependent manner. Expression analyses in melanocytes, melanoma cells, nevi, and primary melanoma revealed that pri-miR-29b2~c levels decrease during melanoma progression. Inactivation of miR-29 in vivo with a miRNA sponge in a rapid melanoma mouse model resulted in accelerated tumor development and decreased overall survival, verifying tumor suppressive potential of miR-29 in melanoma. Through integrated RNA sequencing, target prediction, and functional assays, we identified the transcription factors MAFG and MYBL2 as bona fide miR-29 targets in melanoma. Our findings suggest that attenuation of miR-29b2~c expression promotes melanoma development, at least in part, by derepressing MAFG and MYBL2.

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  1. Version published to 10.3390/cancers13061408
  2. Version published to 10.1101/2020.01.27.922153v3 on bioRxiv
  3. eLife

    ###Reviewer #3

    This study addresses the role of the miR29 micro RNAs in the regulation of melanoma development. Expression of miR29 that is generated from pre-miRs from two clusters is regulated by oncogenic BRAF in human melanocytes. Levels of the mature miR29 are down-regulated in melanoma compared to untransformed melanocytes or nevi and inhibition of miR29 function increases melanoma growth in a murine in vivo model. From RNA-seq data and computational analyses, the authors identify the small MAF protein MAFG as a novel target of miR29 that is involved in melanoma growth. . This study is focused on the function of miR29 in melanoma. The necessity of having the first two figures relevant only for the role of oncogenic BRAF and NRAS in regulating miR29 expression in MEFs is not obvious. Perhaps only one of the two should be shown as a main figure while the other could be moved to the supplemental figures.

    The authors state that TPA regulates the MAPK pathway, but this is misleading as the primary target for TPA is PKC. This should be corrected.

    The comparison of miR29 expression in the collection of melanocyte and melanoma lines uses a poor logic. BRAFV600E expression in primary melanocytes leads to senescence, the HERMES lines are already immortalized by exogenous expression of various genes (CDK4 etc), but this is not mentioned. What would be the effect of BRAFV600E expression in primary melanocytes on Mir29 and MAFG expression? The comparison between these melanocyte lines and the melanoma lines is also misleading as while they all share the BRAFV600E mutation, the melanoma lines have very different transcriptional signatures some being of melanocytic phenotype and other de-differentiated phenotypes. This is not mentioned and how the differences in transcriptional phenotype and P53 status affect miR29 and MAFG expression is not mentioned (see also comment below).

    The description and characterization of the mouse melanoma models is not acceptable as presented. There are no images of tumours, no measure of number and size of tumours or tumour progression only Kaplan-Meier plots of viability. It is impossible for the reader to assess the conclusions from the figure, the additional data should be added. Also, can the authors show that the mouse tumours (or the cells established in vitro) express Mafg and that its levels are altered in the different genetic backgrounds. If not then another mechanism is maybe operative in the mouse tumours.

    The RNA-seq data following expression of the miR29 mimics is not fully described, how many genes were changed, what is fold change of the genes that were subsequently selected for further study, in particular MAFG?

    The changes in MAFG protein expression in Figure 6A are minor. What is the evidence that such small changes can really impact cell growth (see below)? At face value, basal MAFG expression in H1B melanocytes appears higher than in WM164 cells and its levels in H1B cells can only be mildly affected by modulating miR29. Can the authors comment. More importantly, in Figure 6F some highly tumorigenic lines like 1205Lu or SK-Mel-28 have MAFG levels comparable to the HERMES lines. This does not support the authors’ hypothesis that MAFG levels are major regulators of tumorigenic capacity. There is no obvious correlation between the MAFG mRNA and protein levels comparing panels E and F. Also, what are the relative levels of miR29 in these different cell types, do they correlate with MAFG protein levels or are differences in MAFG levels explained by other regulatory mechanisms? Is there any correlation between MAFG protein levels and cell growth rates and clonogenic capacity amongst the different analyzed melanoma lines? Resolving these issues would strengthen the conclusions.

    To fully demonstrate that the effects of miR29 in regulating tumour growth are principally mediated via MAFG, the authors must show they can rescue cell growth defects upon miR29 expression, by expressing MAFG from a cDNA that is insensitive to miR29 regulation. This experiment will help to exclude the implication of other potential miR29 targets in regulation of melanoma cell growth.

  4. eLife

    ###Reviewer #2

    The manuscript by Vera and colleagues dissects the mechanism of miR-29 family expression in melanoma and provides a possible target to support its tumour-suppressive functions. Towards this the expression of miR-29 family upon MAPK and P53 signalling is carefully followed in transgenic mice and humans and classical target analysis is performed. However a few points remain to be addressed:

    Subsection “The MAPK pathway regulates miR-29 expression in human melanocytes and melanoma cells”: "Our results indicate that BrafV600E-induced expression of miR-29 may form a tumour suppressive barrier that restricts the full transformation of melanocytes." This is an overstatement. While the authors clearly show a tumour suppressor role for miR-29 and clearly show that it is induced by MAPK signalling, they never prove that inhibition of miR-29 supports melanocyte transformation.

    Discussion section: "Thus, our work has uncovered that miR-29 prevents melanoma progression downstream of MAPK signalling by repressing MAFG." Again overstatement. Although the authors prove that MAFG is important in melanoma and it is a target of miR-29, they never prove that the activity of miR-29 is mediated by MAFG. A rescue experiment is missing here. The sentence needs a rewording.

    Additionally, the authors could add (expression) correlation analysis between miR-29 and MAFG in human melanoma samples from publicly available databases.

  5. eLife

    ###Reviewer #1

    The goal of this manuscript is appealing. The authors wish to evaluate the importance of mir-29 and MAFG in melanoma progression that would be linked to the activation of the MAPK pathway. This article presents a huge amount of biochemical experiments; it has a potential. However, a significant number of issues must be clarified.

    The MAPK pathway is induced in the very large majority of melanoma, the role of mir29 and MAFG should then be observed in the vast majority of melanomas. Is it the case? If not, what is(are) the main cause(s)? The authors used BRAF V600E, which is perfectly understandable in the case of melanoma, and they also used KRAS G12D. This last mutation is very rare in melanoma. Why not address a similar question with NRAS Q61K/R?

    Choice of the cellular models:

    1. The authors focus on mouse embryonic fibroblasts (MEF) in the two first figures. What is the significance of MEF for human melanoma? Why not using primary melanocytes from human (NHEM) and/or established mouse melanocyte cell lines?

    2. In this study, as models the authors use mouse (MEF and transgenic) and human (melanoma and melanocyte) species. A crucial question is: are the targets for mir29 the same in humans and mice? The conservation of miR and targets is very poor between species. This needs to be addressed.

    3. The authors have to better explain their conclusion of Figure 1. All the presented experiments were performed in MEF. What would happen if the authors used cells from the intestine to evaluate the consequence of BRAFV600E on miR-29? The choice of intestine is not random. What is the link with melanoma? The reason for using mouse embryonic fibroblasts is fine to study molecular issues for this type of cells. However, it is fully accepted that the responses of melanoma to various agents are highly variable.

    Quality of the presented results and reproducibility:

    I will not go through all of the experiments. I will make some remarks.

    1. Figure 1A: The abundance of pERK is moderately induced after expression of KRAS G12D. The authors have to show quantifications on several independent experiments to be convincing.

    2. Figure 3E: The culture media are different in melanocyte and melanoma cell lines. It is therefore difficult to compare the level of miRs. For nevi and melanoma, there is also a pitfall. What is the level of these miR in the stroma? What is the percentage of stromal cells in these biopsies?

    3. Figure 4: There is a clear action of miR-29 sponge in melanoma initiation in mice. What are the targets in mice? According to their models, are they the same in humans? According to the claim of the authors on progression, we expect that the mice have more metastasis? Is it the case? The authors present an overall survival curve. Knowing the ethical rules associated with mouse studies, the authors do not show the survival since they have to sacrifice the mice. The authors have to show the associated raw data.

    4. Figure 7: To further test the importance of MAFG as an oncogene, the authors have to evaluate the growth proliferation in a medium lacking major supplement allowing melanocytes to grow in culture, to reduce the amount of serum, and to test the ability of these cells to grow in 3D or/and in mice.

    Terminologies are vague and/or not defined:

    1. What do the authors refer to "melanoma progression"? In vivo, the authors address the question of melanoma initiation. There is no information on invasion or metastasis. This is crucial according to their title.

    2. AOf course according to the title we wonder if this function is attributed to miR-29a? miR-29b? miR-29c? All? Proper introduction of these three miRs must be done including the known targets of these Mirs. Of course, it has to include the knowledge associated with the different species. In particular, they have to make the point for mouse and humans.

    3. The authors refer to physiological conditions in vitro on plastic in the presence of calf serum. The authors must reformulate the text accordingly and tone down their conclusions.

    4. The authors refer to "full transformation of melanocytes". What do they refer to? It is too vague. Molecular? Cellular?

    Bypass of senescence in melanomagenesis:

    Bypass of senescence is mainly due to the RB/INK4A during melanomagenesis. P53 may be involved but appears to occur later. The authors must address this issue, especially when they use Hermes cells.

    TPA induces mainly PKC and not the MAPK pathway as the authors mention. The authors should clearly show that the MAPK pathway is indeed induced, not only using pERK. Here, in this context, the WB analyses are not sufficient. Moreover, what would be the action of dbcAMP and aMSH?

    Additional comment:

    The authors could present a clear and comprehensive scheme for humans (and mice?) representing the associated pathways.

  6. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to Version 2 of the preprint.

    ###Summary This study addresses the role of the miR29 microRNAs in the regulation of melanoma development. Expression of miR29 that is generated from pre-miRs from two clusters is regulated by oncogenic BRAF in human melanocytes. Levels of the mature miR29 are down-regulated in melanoma compared to untransformed melanocytes or nevi and inhibition of miR29 function increases melanoma growth in a murine in vivo model. From RNA-seq data and computational analyses, the authors identify the small MAF protein MAFG as a novel target of miR29 that is involved in melanoma growth.

    We found this study interesting, but we are of the opinion that the central hypothesis that miR29 regulates MAFG levels to influence melanoma is not yet fully substantiated by the data. Critical experiments could be added, for example, the rescue of growth defects upon miR29 mimic expression with a miR-insensitive form of MAFG, or evidence that Mir29 regulation of Mafg is involved in the mouse melanoma. Furthermore, we do not feel that the immunoblots support the idea that MAFG promotes tumour growth as the 1205LU cells that are highly tumorigenic in nude mice have MAFG levels comparable to the melanocytes lines.

  7. Version published to 10.1101/2020.01.27.922153v2 on bioRxiv
  8. Version published to 10.1101/2020.01.27.922153v1 on bioRxiv