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A non-coding function of TYRP1 mRNA promotes melanoma growth

Abstract

Competition among RNAs to bind miRNA is proposed to influence biological systems. However, the role of this competition in disease onset is unclear. Here, we report that TYRP1 mRNA, in addition to encoding tyrosinase-related protein 1 (TYRP1), indirectly promotes cell proliferation by sequestering miR-16 on non-canonical miRNA response elements. Consequently, the sequestered miR-16 is no longer able to repress its mRNA targets, such as RAB17, which is involved in melanoma cell proliferation and tumour growth. Restoration of miR-16 tumour-suppressor function can be achieved in vitro by silencing TYRP1 or increasing miR-16 expression. Importantly, TYRP1-dependent miR-16 sequestration can also be overcome in vivo by using small oligonucleotides that mask miR-16-binding sites on TYRP1 mRNA. Together, our findings assign a pathogenic non-coding function to TYRP1 mRNA and highlight miRNA displacement as a promising targeted therapeutic approach for melanoma.

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Figure 1: TYRP1 mRNA drives melanoma growth.
Figure 2: TYRP1 mRNA sequesters miR-16.
Figure 3: Biological and clinical significance of the TYRP1 mRNA as a miRNA sponge.
Figure 4: RAB17 promotes proliferation of melanoma cells and in vivo tumour growth.
Figure 5: Target site blocker restores miR-16 function.
Figure 6: Restoring miR-16 tumour-suppressor function reduces tumour growth.

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Acknowledgements

The authors thank the Gene Expression and Oncogenesis team, especially K. Tutoré, for helpful discussions; N. Cougot, R. Gillet, A. Méreau and E. Giudice from the CNRS UMR6290; the Rennes FHU CAMIn team; and B. Turlin, J. Le Seyec, M. Trotard and A. Popa for providing scientific expertise. The authors acknowledge the SFR Biosit core facilities of Rennes University with the histopathology H2P2 (A. Fautrel) and cell imaging ImPACcell (R. Le Guevel) platforms, and the ARCHE animal housing facility for their help and support. The authors also thank the Genomic platform from the translational research department of the Curie Institute in Paris. This study received financial support from the following: Institut National du Cancer PAIR Melanoma program; Ouest Valorisation; Ligue National Contre le Cancer (LNCC) Départements du Grand-Ouest; Région Bretagne; University of Rennes 1; CNRS; Association Transfusion Sanguine et Biogénétique Gaétan Saleun; MEDIC Foundation; Les Amis de l’Institut Bordet; Fondation Lambeau-Marteaux; European Organisation for Research and Treatment of Cancer Tournesol program; and the Société Française de Dermatologie. Further support was provided by a ‘Ligue Nationale Contre le Cancer’ (LNCC) fellowship (E.D.-F.) and from the Région Bretagne and the LNCC Grand Ouest (A.G.; M.M.) and from Faculté des Sciences Pharmaceutiques de l’Université de Rennes 1 (M.M.). The authors are grateful to G. Lizée for providing the Mel624 cell line, to R. B. Darnell for providing Huh7.5 Drosha KO cells, to J. Wrana for pMS2 vectors and to V. J. Hearing for the anti-TYRP1 (PEP1) antibody.

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Conceptualization: D.G., M.M. and M.-D.G.; methodology: D.G., M.M., L.B., A.R., A.M., N.M., M.-L.P.-M., T.M., S.C., B.F. and S.T.-D.; investigation: D.G., M.M., L.B., F.J., A.R., E.D.-F., A.M., N.M., M.-L.P.-M., T.M., S.C., A.G. and R.B.J.; formal analysis: D.G., M.M., F.J., A.R., N.M., B.M. and F.R.; writing—original draft: D.G., M.M. and M.-D.G.; funding acquisition: D.G., F.J., T.M., S.C., G.G. and M.-D.G.; resources: F.J., F.R., P.E., J.-C.M. and G.G.; supervision: D.G. and M.-D.G.

Corresponding authors

Correspondence to David Gilot or Marie-Dominique Galibert.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Moderate TYRP1 mRNA knockdown reduces melanoma cell proliferation.

(a,b) Proliferation rate of shCTR and shTYRP1 (moderate KD) 501Mel (a) or ME1402 cells (b). Cells were counted every two days during eight or ten days (n = 2 biologically independent experiments in triplicate). TYRP1 protein was detected by Western blot experiments; pictures are representative of three independent experiments. HSC70 serves as loading control. (c,d) TYRP1 knockdown in two melanoma short-term cultures (MM057 & MM165) and in Mel624 melanoma cell line. Cell density (c) and TYRP1 mRNA levels (d) have been evaluated 4 or 7 days after infection (shTYRP1) or transfection (siTYRP1). N.D. for not detected. Each histogram represents the mean of 2 or 3 biologically independent experiments (n = 2 for cell density experiments; n = 2 for MM057 & Mel624 and n = 3 for MM165 for TYRP1 mRNA level quantification). (e) TYRP1 knockdown in two melanoma cell lines expressing TYRP1 mRNA. Three different antibodies (PEP1, AB23 & G17) were used to confirm the absence of the TYRP1 protein in SKMel28-luc cells. Pictures are representative of three independent experiments. Source data are available in Supplementary Table 8 and unprocessed original blots are shown in Supplementary Fig. 7.

Supplementary Figure 2 miR-16 reduces TYRP1 mRNA decay induced by miR-155.

(a) Cartoon illustrating the 3′UTR of human TYRP1 with the position of the SNPs rs683 and rs910, the three MRE-155 sites (blue) and the three putative MRE-16 sites (orange). (b) MRE-155 sequences on TYRP1 3′UTR. TYRP1-C corresponds to the NM_000550.2 and TYRP1-A to NM_000550.1. Alignments have been performed using RNAhybrid34. Underlined sequences on MRE-155#2C and MRE-155#3A are detailed in boxes on the right to show the position of the SNPs in the two alleles of TYRP1. Arrows indicate the SNP rs683 and rs910 positions. (c) Effects of synthetic miR-155 on the identified regions of TYRP1 MRE-155 in 501Mel. Luciferase activity was evaluated 48 h after transfection. Each histogram represents the mean ± s.d. of n = 3 biologically independent experiments; two-sided unpaired t-test with Welch’s correction; P < 0.05. (d) Northern blot quantification of miR-16 in 501Mel cells. The signal (from 501Mel cells) was fit to the standard curve from synthetic titration signals to give final copy number per cell. RNU6 served as a loading control. Pictures are representative of three experiments. (e) Northern blot quantification of TYRP1 mRNA in 501Mel cells. The signal (from 501Mel cells) was fit to the standard curve from the TYRP1 3′UTR’s synthetic titration signal to give final copy number per cell. GAPDH serves as a loading control. Picture presents three biological replicates of 501Mel. Source data are available in Supplementary Table 8.

Supplementary Figure 3 MRE-16 on human and mouse TYRP1 mRNA and biological consequences.

(a) MRE-16s’ sequences on mouse TYRP1 3′UTR (NM_031202.3). Alignments have been performed using RNAhybrid34. (b) Schematic representation of the interaction (purple base paring) of miR-16 (orange) and miR-155 (blue) with human and mouse TYRP1 MRE-16#3 and MRE-155#3 respectively. (c,d) TYRP1 knockdown in mouse B16-F10 melanoma cells using three different siRNAs. TYRP1 mRNA levels (c) and cell density (d) have been respectively evaluated at 5 and 3 days after siRNA transfection; one-way ANOVA with Holm-Sidak’s multiple comparisons test. (e) Effect on cell density of synthetic miR-16 transfected in mouse B16-F10 melanoma cells 3 days after miRNA transfection; two-sided unpaired t-test with Welch’s correction, P < 0.05. Each histogram represents the mean ± s.d. (n = 3 biologically independent experiments). Source data are available in Supplementary Table 8.

Supplementary Figure 4 TYRP1 silencing decreases expression level of several mRNAs.

(a) Workflow to identify deregulated RNAs in TYRP1 KD cells. Gene expression profile of cells transfected with three different siRNAs targeting TYRP1 or siRNA CTR. Significance analysis of microarrays was done as described in methods. siRNA efficacy for TYRP1 KD is #2 > #3 > #1. Heatmap focused on deregulated RNAs in function of siTYRP1 efficacy (top) and a z-score has been calculated using Ingenuity Pathway Analysis (IPA) (bottom). (b) mRNA expression levels of TYRP1, MAFF, NRTN, RAB17 and RasGRP3 in response to TYRP1 KD using three different siRNAs (#1-3) targeting the ORF of TYRP1 in 501Mel cells (n = 4 biologically independent experiments except for RASGRP3 expression measurement which results from n = 3 biologically independent experiments). (c) mRNA expression levels of TYRP1, MAFF, NRTN, RAB17 and RasGRP3 in response to TYRP1 KD using shTYRP1 targeting the ORF of TYRP1 in SKMel28-luc cells (n = 3 biologically independent experiments). (d) Cell density of 501Mel cells in response to siRNAs targeting MAFF, NRTN, RAB17 or RasGRP3 (n = 3 biologically independent experiments; one-way ANOVA with Holm-Sidak’s multiple comparisons test, P < 0.05). Two siRNAs were used by target. (e) mRNA expression levels in response to synthetic miR-16. Each histogram represents the mean ± s.d. (n = 3 biologically independent experiments). TYRP1 and RAB17 mRNA expression in response to synthetic miR-16 are reported in Figs 2d and 4b, respectively. (f) The MRE-16s’ sequence on human MAFF and NRTN mRNAs have been identified using RNAhybrid34. For b,c and e, two-sided unpaired t-tests with Welch’s correction were done (P < 0.05). For be, values correspond to the mean ± s.d. Source data are available in Supplementary Table 8.

Supplementary Figure 5 Overall survival of patients with metastatic melanoma according to TYRP1, RAB17, NRTN mRNAs or miR-16 expression levels.

(a,b) Determination of overall survival (OS) curves by Kaplan–Meier analysis, according to the expression levels of RAB17 (a) or TYRP1 and RAB17 (b). Based on 184 skin and lymph node metastases from melanoma patients (IJB cohort). High and low expression of TYRP1 and RAB17 (a) alone were defined based on their median values and scored as 1 and 0, respectively. For combination (b), scores of TYRP1 (1 or 0) and RAB17 (1 or 0) are added and the resulting scores 1 and 2 are combined as the high score (n = 130 patients), which was significantly different from the low score 0 (n = 54 patients) regarding to OS values (two-sided Mann–Whitney test, P = 0.004). Cox regression was used to calculate P values, hazard ratios (HR) and 95% confidence intervals (CI). (c) Association of TYRP1 and RAB17 expression with patient survival. TYRP1 and RAB17 expression levels were assessed in the TCGA SKCM melanoma cohort. Patients were ranked decreasingly according to TYRP1 and RAB17 expression, resulting in almost three equal groups. The Kaplan-Meier curve representing the highest third of TYRP1 and RAB17 expressers shows a significantly lower OS as compared to the lowest third (log-rank test). (d,e) Determination of OS curves by Kaplan–Meier analysis, according to the expression levels of NRTN (d) or TYRP1 and NRTN (e). Patients were ranked decreasingly according to NRTN (d) or TYRP1 and NRTN expression (e), resulting in almost four equal groups. The Kaplan-Meier curve represents the highest and the lower quarters of expressers (log-rank test). (f,g) Determination of OS curves by Kaplan-Meier analysis, according to the expression levels of miR-16 from n = 85 patients of the IJB cohort (f) or from n = 349 patients from the TCGA cohort (g). High and low expression groups of miR-16 were defined based on its median value (log-rank test).

Supplementary Figure 6 Histological analyses of liver and kidney from PDX mice and long-term tumor growth.

(a) Representative micrographs of liver and kidney slices stained with hematoxylin from PDX-mice exposed to TSB-C1 or TSB-T3. Toxicity evaluation was blindly examined by two independent pathologists. Two mice have been showed per group among five mice analysed given similar results. Scale bar, 100 μm. (b) Tumor volume for individual PDX mice treated with TSB-C1 or TSB-T3 as described in Fig. 6b. (c) Quantification of LAMP2 mRNA in melanoma tumors treated with TSB-C1 or TSB-T3 (n = 5 mice per group). LAMP2 is not a miR-16 target. For c, lines represent the mean ± s.d.; values represent fold change relative to the mean of TSB-C1 condition; two-sided unpaired t-tests with Welch’s correction; NS, non-significant. Source data are available in Supplementary Table 8.

Supplementary Figure 7 Unprocessed original scans of blots.

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Gilot, D., Migault, M., Bachelot, L. et al. A non-coding function of TYRP1 mRNA promotes melanoma growth. Nat Cell Biol 19, 1348–1357 (2017). https://doi.org/10.1038/ncb3623

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