Palmitoylation-dependent activation of MC1R prevents melanomagenesis

Abstract

The melanocortin-1 receptor (MC1R), a G-protein-coupled receptor, has a crucial role in human and mouse pigmentation1,2,3,4,5,6,7,8. Activation of MC1R in melanocytes by α-melanocyte-stimulating hormone (α-MSH)9 stimulates cAMP signalling and melanin production and enhances DNA repair after ultraviolet irradiation10,11,12,13,14,15,16. Individuals carrying MC1R variants, especially those associated with red hair colour, fair skin and poor tanning ability (denoted as RHC variants), are associated with higher risk of melanoma5,17,18,19,20. However, how MC1R activity is modulated by ultraviolet irradiation, why individuals with red hair are more prone to developing melanoma, and whether the activity of RHC variants might be restored for therapeutic benefit are unknown. Here we demonstrate a potential MC1R-targeted intervention strategy in mice to rescue loss-of-function MC1R in MC1R RHC variants for therapeutic benefit by activating MC1R protein palmitoylation. MC1R palmitoylation, primarily mediated by the protein-acyl transferase ZDHHC13, is essential for activating MC1R signalling, which triggers increased pigmentation, ultraviolet-B-induced G1-like cell cycle arrest and control of senescence and melanomagenesis in vitro and in vivo. Using C57BL/6J-Mc1re/eJ mice, in which endogenous MC1R is prematurely terminated, expressing Mc1r RHC variants, we show that pharmacological activation of palmitoylation rescues the defects of Mc1r RHC variants and prevents melanomagenesis. The results highlight a central role for MC1R palmitoylation in pigmentation and protection against melanoma.

Figure 1: Palmitoylation of MC1R in melanocytes.
Figure 2: ZDHHC13 is a major MC1R palmitoyl acyltransferase.
Figure 3: Activating MC1R palmitoylation rescues the defect of MC1R RHC variants.
Figure 4: MC1R palmitoylation controls melanomagenesis.

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Acknowledgements

We thank X. Yao, H. Xie and G. Wei for careful reading and suggestions. This work was supported by the National Institutes of Health (R.C.: R01CA137098, R01CA193913 and R01CA196896), Department of Defense (R.C., CA140020), Melanoma Research Foundation Establish Investigator Award (R.C.), Hong Kong and Macao Young Scientists of the National Natural Science Foundation of China (R.C., 81428025), National Natural Science Foundation of China (X.G., 81630106), and the Ludwig Institute for Cancer Research (C.R.G.). R.C. is an American Cancer Society Research Scholar.

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Contributions

R.C. conceived the hypothesis, organized and supervised the study. R.C. designed the project with help from S.C., B.Z. and C.Y. S.C., B.Z. and C.Y. analysed and interpreted data. S.C. performed mouse experiments (melanomafree survival, histopathology) with assistance from C.Y. B.Z. independently reproduced mouse experiments with assistance from W.L. and C.H. S.C. performed protein palmitoylation assays. B.Z. and C.Y. independently reproduced protein palmitoylation assays with assistance from W.L. and C.H. S.C. performed melanocytes function (cAMP, MITF, DNA repair, senescence) measurement and cellular transformation assay, which were independently reproduced by C.Y. B.C. and X.W. designed and performed the transfection of HA–ZDHHC, which was independently reproduced by S.C. and C.Y. T.L., X.L., X.C., C.L., L.H., J.Z., Z.X., X.G. and C.R.G. contributed to data analysis, interpretation and revision of the manuscript. R.C. wrote the manuscript with help from C.R.G. All authors commented on the manuscript.

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Correspondence to Rutao Cui.

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Reviewer Information Nature thanks G. Merlino and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 MC1R is palmitoylated.

a, B16-RHC cells exposed to α-MSH and UVB-irradiated were treated with BSA-conjugated fatty acids. b, A schematic showing the general process of protein palmitoylation. c, B16-RHC cells were exposed to palmitic acid ± 2-BP. d, e, MC1R RHC-variant or wild-type B16 melanoma cells (d) and MC1R RHC-variant or wild-type HPMs (e) were treated as indicated in Fig. 1a or ± 2-BP. cAMP were calculated by three independent experiments, shown as mean ± s.d. **P < 0.01, ***P < 0.001, unpaired Student’s t-test. f, A schematic showing the general process of the acyl-biotin exchange (ABE) palmitoylation assay. g, Flowchart of palmitoylated protein identification. NEM, N-ethylmaleimide; HAM, hydroxylamine. h, The peptide spectral counts of MC1R from Fig. 1c. i, Palmitoylation site prediction (NBA-palm) analysis of MC1R. The prediction shows two possible sites of MC1R palmitoylation. j, A schematic illustration showing the palmitoylation site at MC1R(C315) predicted by palmitoylation site prediction analysis and PEP-FOLD3, and a schematic showing the conserved C-terminal domain of MC1R. k, Membrane topology of MC1R with indicated RHC, non-RHC and palmitoylation site mutants. Source data

Extended Data Figure 2 Palmitoylation of MC1R in melanocytes.

af, B16 cells (a), B16 cells expressing wild-type, mutant or variant Flag–MC1R (be), HPMs expressing wild-type, mutant or variant Flag–MC1R as indicated (f) were incubated with α-MSH and processed for ABE analysis. gm, B16 cells (g), B16 cells expressing wild-type, mutant or variant Flag–MC1R (hk, m), HPMs expressing wild-type, mutant or variant Flag–MC1R (l) were treated with α-MSH, irradiated with UVB, and collected for ABE analysis. Western blots shown are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 3 ZDHHC13 is a major protein S-acyl transferase of MC1R.

a, HEK293 cells co-expressing Flag–MC1R and HA–ZDHHCs were collected for ABE analysis. b, B16 cells infected with Flag–MC1R and the indicated wild-type ZDHHC13- or mutant C456S-encoding retroviral constructs were treated with α-MSH and processed for ABE analysis. c, B16 cells expressing Zdhhc13-targeting shRNAs were treated with α-MSH, irradiated with UVB, and collected for ABE analysis. d, B16 cells expressing Flag–MC1R together with Zdhhc13-targeting shRNAs and/or wild-type HA–ZDHHC13 were treated with α-MSH, irradiated with UVB, and collected for ABE analysis. e, f, B16 cells or HPMs with stable MC1R depletion were infected with the Flag–MC1R(R151C)- or MC1R(R151C/C315S)-double-mutant-encoding retroviral constructs, then cells were infected with wild-type HA–ZDHHC13 expressing virus. Finally, cells were treated with α-MSH, irradiated with UVB and processed for ABE analysis. g, h, HPMs pre-incubated with α-MSH followed by UVB irradiation were collected for ABE (g) or immunoprecipitation (h) analysis. i, A schematic showing the conserved SQ motif of ZDHHC13. j, HPMs expressing wild-type ZDHHC13 or ZDHHC13(S8A) mutant were irradiated with UVB and collected for immunoprecipitation and immunoblot analysis. k, Wild-type Flag–ATR or the kinase-dead (KD) Flag–ATR mutant transfected HEK293 cells were irradiated before immunoprecipitation with Flag beads. The immunoprecipitated wild-type ZDHHC13 or S8A mutant were then incubated with immunoprecipitated wild-type ATR or the kinase-dead ATR mutant in kinase buffer. After reaction, proteins were collected for IB analysis. l, HPMs expressing wild-type ZDHHC13 or ZDHHC13(S8A) mutant and Flag–MC1R were irradiated with UVB and collected for immunoprecipitation analysis. m, HPMs with stable depletion of ZDHHC13 by shRNA were infected with the indicated ZDHHC13- and Flag–MC1R-encoding retroviral constructs. Cells were then treated with α-MSH, irradiated with UVB and processed for ABE analysis. Western blots shown are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 4 Palmitoylation is essential for MC1R function.

ac, B16 cells and HPMs expressing the indicated Flag–MC1R were treated with α-MSH and irradiated with UVB. Cells were collected for cAMP immunoassay (a) or quantitative PCR (qPCR) by specific primers targeting mouse/human MITF or TYR (b, c). Data are represented as mean ± s.d. Three independent experiments were quantified. d, HPMs expressing indicated Flag–MC1R were treated with α-MSH and irradiated with UVB. Genomic DNA was extracted at the different time points as indicated, and the photoproducts were detected by ELISA. Three independent experiments were measured and shown as mean ± s.d. e, f, B16 and HPMs with stable depletion of MC1R by shRNA (e), or B16 cells and HPMs expressing indicated Flag–MC1R (f) were pre-treated with α-MSH for 30 min followed by 25 J m−2 UVB irradiation. Cells were subjected to SA-β-gal staining assay 7 days after UVR. Data are represented as mean ± s.d. from three independent experiments. gk, hTERT/p53DD/CDK4(R24C)/BRAF(V600E) melanocytes with stable depletion of MC1R by shRNA (gi) or expressing the indicated Flag–MC1R (j, k) were pre-incubated with 1 μM α-MSH for 30 min before being irradiated with 20 J m−2 UVB. Cell lysates were collected for immunoblot analysis (g, j), or cells were seeded for clonogenic survival (h), and soft agar assays (i, k). Results were calculated as mean ± s.d. from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t-test. Western blots shown are representative of three independent experiments. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Figure 5 Activating MC1R palmitoylation rescues the defect of MC1R RHC variants.

a, b, B16 cells and HPMs expressing Flag–MC1R together with Zdhhc13-targeting shRNAs and/or wild-type HA–ZDHHC13 were treated with α-MSH and irradiated with UVB. Cells were collected for cAMP immunoassay (a) or quantitative PCR (qPCR) by specific primers targeting mouse/human MITF (b). Data are represented as mean ± s.d. Three independent experiments were quantified. c, HPMs expressing Flag–MC1R together with ZDHHC13-targeting shRNAs and/or wild-type HA–ZDHHC13 were treated with α-MSH, irradiated with UVB. Genomic DNA were extracted at the different time points as indicated and photoproducts were detected by ELISA. Three independent experiments were measured and data are represented as mean ± s.d. d, B16 cells and HPMs expressing Flag–MC1R together with Zdhhc13-targeting shRNAs and/or wild-type HA–ZDHHC13 were pre-treated with 1 μM α-MSH for 30 min followed by 25 J m−2 UVB irradiation. Cells were subjected to SA-β-gal staining assay 7 days after UVR. Data are represented as mean ± s.d. from three independent experiments. e, The cells generated as indicated were subjected for soft agar assay and relative colony numbers were plotted as mean ± s.d. from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t-test. Source data

Extended Data Figure 6 MC1R variant and mutant transgenic mice.

a, Schematic diagrams of MC1R variant constructs. Transgenic mice were designed to express melanocyte-specific MC1R variants or mutants (controlled by the Tyr enhancer/promoter). b, C57BL/6 MC1R variant or mutant transgenic mice. c, Human transgene content in transgenic and control mice. Results were calculated as mean ± s.d. from three independent experiments. d, Whole skins from C57BL/6 MC1R variant transgenic mice (8–12 weeks) were collected and stained with Dct antibody. Melanocytes were then isolated and quantified by FACS sorting. Results were calculated as mean ± s.d. from three independent experiments. e, Frozen sections of skins from C57BL/6 MC1R variant transgenic mice (8–12 weeks) were stained with Dct antibody. The positive staining represents melanocytes. f, Illustrations for UVB-induced melanoma development in Tyr-Cre-BRAFCA-MC1R variant mice. g, H&E staining of histological sections and immunohistochemistry staining of S100 of representative cutaneous melanomas. Genotypes are indicated. Source data

Extended Data Figure 7 Palm-B activates MC1R palmitoylation and rescues the defect of MC1R RHC variants.

a, HPMs expressing Flag–MC1R were incubated with α-MSH for 3.5 h. The medium was replaced with fresh medium containing vehicle or 1 μM Palm-B, and cells were treated at the indicated times. Cells were then collected for ABE analysis. bf, B16 cells or HPMs expressing the indicated Flag–MC1R were treated with α-MSH and Palm-B and irradiated with UVB. Cells were processed by ABE analysis (b), cAMP immunoassay (c), qPCR (d, e) and photoproducts were measurement (f). Three independent experiments were quantified. Data are represented as mean ± s.d. g, C57BL/6 mice or C57BL/6-Mc1re/eMc1rR151C-transgenic mice were given a 10 mg kg−1 Palm-B or vehicle injection intraperitoneally 3 h before UVB irradiation (500 J m−2). 3 h after UVB, whole skins were collected and the lysates were subjected for ABE analysis. h, i, C57BL/6 mice or C57BL/6-Mc1re/eMc1rR151C-transgenic mouse were injected intraperitoneally with 10 mg kg−1 Palm-B 3 h before UVB irradiation (500 J m−2). Melanocytes were isolated by flow cytometry, then DNA was extracted and subjected to ELISA 3 h after UVB irradiation. Results were calculated as mean ± s.d. from three independent experiments. j, B16 cells and HPMs expressing the indicated Flag–MC1R were pre-treated with α-MSH and Palm-B for 30 min followed by 25 J m−2 UVB irradiation. Cells were subjected to SA-β-gal staining assay 7 days after UVR. Data are represented as mean ± s.d. from three independent experiments. k, The cells generated as indicated were subjected for soft agar assay and relative colony numbers were plotted as mean ± s.d. from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t-test. Western blots shown are representative of three independent experiments. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Figure 8 Palm-B rescues the defect of MC1R R160W variant.

ad, B16 cells or HPMs expressing the indicated Flag–MC1R were treated with α-MSH and Palm-B and irradiated with UVB. Cells were processed by ABE analysis (a), cAMP immunoassay (b), qPCR (c) and photoproducts were measurement (d) (three independent experiments). Data are represented as mean ± s.d. e, B16 cells and HPMs expressing the indicated Flag–MC1R were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 25 J m–2 UVB irradiation. Cells were subjected to SA-β-gal staining assay 7 days after UVR. Data are represented as mean ± s.d. from three independent experiments. f, g, MC1R-depleted TERT/p53DD/CDK4(R24C)/BRAF(V600E) melanocytes expressing indicated Flag–MC1R were pre-incubated with 1 μM α-MSH and 1 μM Palm-B for 30 min before being irradiated with 20 J m−2 UVB, and then subjected to clonogenic survival (f) and soft agar assay (g). Results were shown as mean ± s.d. from three independent experiments. **P < 0.01, ***P < 0.001, unpaired Student’s t-test. Western blots shown are representative of three independent experiments. For gel source data, see Supplementary Fig. 1. Source data

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Chen, S., Zhu, B., Yin, C. et al. Palmitoylation-dependent activation of MC1R prevents melanomagenesis. Nature 549, 399–403 (2017). https://doi.org/10.1038/nature23887

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