Ras-Induced miR-146a and 193a Target Jmjd6 to Regulate Melanoma Progression

Abstract

Ras genes are among the most commonly mutated genes in human cancer; yet our understanding of their oncogenic activity at the molecular mechanistic level is incomplete. To identify downstream events that mediate ras-induced cellular transformation in vivo, we analyzed global microRNA expression in three different models of Ras-induction and tumor formation in zebrafish. Six microRNAs were found increased in Ras-induced melanoma, glioma and in an inducible model of ubiquitous Ras expression. The upregulation of the microRNAs depended on the activation of the ERK and AKT pathways and to a lesser extent, on mTOR signaling. Two Ras-induced microRNAs (miR-146a and 193a) target Jmjd6, inducing downregulation of its mRNA and protein levels at the onset of Ras expression during melanoma development. However, at later stages of melanoma progression, jmjd6 levels were found elevated. The dynamic of Jmjd6 levels during progression of melanoma in the zebrafish model suggests that upregulation of the microRNAs targeting Jmjd6 may be part of an anti-cancer response. Indeed, triple transgenic fish engineered to express a microRNA-resistant Jmjd6 from the onset of melanoma have increased tumor burden, higher infiltration of leukocytes and shorter melanoma-free survival. Increased JMJD6 expression is found in several human cancers, including melanoma, suggesting that the up-regulation of Jmjd6 is a critical event in tumor progression. The following link has been created to allow review of record GSE37015: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jjcrbiuicyyqgpc&acc=GSE37015.

Keywords: Jmjd6; cancer models; melanoma; miR-146a; miR-193a; microRNA; ras; zebrafish.

FIGURE 1

miR-21, miR-146a/b, and miR-193a are upregulated by oncogenic RAS in transgenic cancer models. (A) Zebrafish models used in the study of Ras-dependent microRNAs. Green fluorescence denotes the expression of the eGFP-fused oncogene. In Kita:Ras GFP labels transformed melanocytes and notochord; in zic:Ras GFP marks ras-expressing brain cells; in HS-Ras, eGFP-Ras is expressed in whole embryos. For full description of transgenic lines, see text. (B) Diagram depicting the overlap between the three sets of upregulated microRNAs. (C) Taqman QPCR analysis of miR-21, miR-146a/b, and miR-193a expression levels in 3 dpf kita-RAS larvae compared to control (CT) larvae. The error bar represents the SEM of a triplicate experiment. (D) Heatmap representation of microarray analysis of miR-21, miR-146a/b, and miR-193a expression at different stages of melanoma progression. Distinct precursor sequences and genomic loci that express identical mature sequences are named on the form miR-21-1 and miR-21-2. Lettered suffixes denote closely related mature sequences. –5p and –3p indicate the 5′ and 3′ arm respectively. ^∗P ≤ 0.05 and ^∗∗P ≤ 0.01.

FIGURE 2

The increase of miR-21, miR-146a/b, and miR-193a is RAS-dependent. (A) Taqman QPCR analysis of miR-21, 146a, 146b, and 193a expression following ras upregulation (6 h after heat-shock induction), ^∗p < 0.05. (B) Diagram showing the ras pathway inhibitors used and their targets. (C) Western Blot analysis of ERK-P, AKT-P, and S6-P ribosomal protein levels after the inhibitors treatment. (D–G) Taqman qPCR analysis in 3 dpf HS-RAS zebrafish treated with PD98059, LY29004 and rapamycin. The error bars represent the SEM of a triplicate experiment; two tailed Student’s test was used for analysis. ^∗p < 0.05.

FIGURE 3

Jmjd6 is a target of miR-146a and miR-193a. (A) Effects of increasing microRNA levels on a Jmjd6-GFP sensor expression. Diagram of the construct used as sensor. (B) Representative images of 24 hpf zebrafish embryos injected with the Jmjd6-3′ UTR GFP sensor and the microRNA duplexes as indicated. Arrows illustrate the direction of the changes in expression. (C) Western blot analysis of GFP expression and (D) quantification of the changes in Jmjd6-3′ UTR GFP sensor levels upon microRNA overexpression. (E) Reduction of Jmjd6 protein levels following miR-146a, and miR-193a duplex injection in 3 dpf embryos shown by Western Blot analysis. (F) Quantification of the Western Blot shown in (E). (G) RIP (RNA immuno precipitation) diagram and (H) analysis of jmjd6 transcripts in the RISC complex following duplex injections in 3 dpf embryos.

FIGURE 4

JMJD6 is up-regulated in zebrafish and human melanoma. (A) Jmjd6 mRNA levels in 3, 7, 14 days kita-RAS zebrafish and (B) in adult melanoma developing in two different genetic backgrounds, as shown. The error bars represent the SEM of triplicate experiments; two tailed Student’s test was used for analysis. ^∗p < 0.01. (C) Analysis of JMJD6 expression in a variety of cancers from the cBioPortal database; every spot represents a case. (D) Expression of JMJD6 (brown nuclear staining) in control skin and in a malignant melanoma from the Protein Atlas website (https://www.proteinatlas.org/ENSG00000070495-JMJD6).

FIGURE 5

Expression of miR-resistant Jmjd6 promotes Ras-induced melanoma. (A) Schematic representation of the construct used to generate a transgenic line expressing microRNA-resistant Jmjd6 under the UAS promoter. Cmlc2-GFP is the cardiac myosin light chain promoter driving GFP expression in the heart as marker of transgenesis. (B–D) Examples of GFP staining in crosses between the UAS lines and the kita:Gal4 line (as indicated). Expression is visible in the notochord (Distel et al., 2009) and in melanocytes in all kita crosses (B–D), and in the heart for the UAS:Jmjd6 line, C,D) GFP is localized to the nucleus for Jmjd6-GFP or the plasma membrane for eGFP-HRASV12. (E) Double or triple transgenic larvae and juveniles at the indicated stages of development. (F) Disease- free survival curve of the double or triple transgenic fish. N = 100 for kita; 321 for kita:Ras; 211 for kita:Ras:Jmjd6. Long-rank test: p-value < 0,0001. (G,H) H&E staining of representative melanoma sections from transgenic fish as indicated. (I–L) Immunostaining for L-plastin (red florescence) and GFP (only in I–K, indicating HRASV12, green fluorescence) in representative melanoma sections from transgenic fish as indicated. Calibration bar: 100 μm for (B,D), 50 μm for (I–L).