Abstract
The BRAF kinase is mutated, typically Val 600→Glu (V600E), to induce an active oncogenic state in a large fraction of melanomas, thyroid cancers, hairy cell leukaemias and, to a smaller extent, a wide spectrum of other cancers1,2. BRAFV600Ephosphorylates and activates the MEK1 and MEK2 kinases, which in turn phosphorylate and activate the ERK1 and ERK2 kinases, stimulating the mitogen-activated protein kinase (MAPK) pathway to promote cancer3. Targeting MEK1/2 is proving to be an important therapeutic strategy, given that a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma4, an effect that is increased when administered together with a BRAFV600Einhibitor5. We previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu–MEK1 interaction6. Here we show decreasing the levels of CTR1 (Cu transporter 1), or mutations in MEK1 that disrupt Cu binding, decreased BRAFV600E-driven signalling and tumorigenesis in mice and human cell settings. Conversely, a MEK1–MEK5 chimaera that phosphorylated ERK1/2 independently of Cu or an active ERK2 restored the tumour growth of murine cells lacking Ctr1. Cu chelators used in the treatment of Wilson disease7decreased tumour growth of human or murine cells transformed by BRAFV600E or engineered to be resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat cancers containing the BRAFV600Emutation.
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Acknowledgements
We thank M. McMahon, C. Cronin, E. Johnson, A. Stewart, D. S. Tyler and D. G. Kirsch for reagents, and C. Cronin, L. E. Crose, A. M. Jaeger, M. A. Luftig, E. Johnson, D. F. Kashatus, B. L. Lampson, J. P. Madigan, N. I. Nicely, Y. Nose, C. W. Pemble, N. L. K. Pershing, A. Stewart and J. D. Weyandt for technical support, discussions and/or review of the manuscript. This work was supported by National Institutes of Health grants CA178145 (D.C.B.), HL075443 (Proteomic Core K.X.), DK074192 (D.J.T.), CA094184 and CA172104 (C.M.C.), the Structural Genomics Consortium (Welcome Trust 092809/Z/10/Z), FP7 grant 278568 ‘PRIMES’ (S.K. and A.C.), the Stewart Trust (C.M.C.), the Edward Spiegel Fund of the Lymphoma Foundation (C.M.C.), and donations made in memory of Linda Woolfenden (C.M.C.).
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Experiments were performed by D.C.B., M.S.C., M.L.T., G.A.H., X.Y. and K.X. All authors contributed to the study design. The manuscript was written by D.C.B. and C.M.C. with contributions by all authors.
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A provisional patent application (D.C.B., M.L.T., D.J.T. and C.M.C.) has been filed regarding the use of Cu chelators for the treatment of BRAF and RAS mutation-positive cancers and a phase I melanoma trial investigating a copper chelator has been registered with clinicaltrials.gov (NCT02068079) with one of the authors serving as a co-investigator (C.M.C.).
Extended data figures and tables
Extended Data Figure 1 CuSO4stimulates MEK1/2 kinase activity in vitro.
a, c, Detection of the amount of in vitro phosphorylated (P) recombinant GST-tagged kinase-inactive ERK2K54R protein by recombinant GST-tagged MEK1 in the presence, when indicated, of 2.5 molar equivalents of CuSO4 (Cu), AgNO3 (Ag), FeNH2SO4 (Fe), NiSO4 (Ni) or ZnSO4 (Zn) (a) or recombinant GST-tagged MEK2 in the presence, when indicated, of 2.5 μM CuSO4 and/or 50 μM TTM (c). Total (T) levels of ERK2, MEK1 and MEK2 serve as loading controls. b, Immunoblot detection of the amount of recombinant GST-tagged MEK2 protein bound to a resin charged with (Cu) or without (−) Cu. Input serves as a loading control. Gel images are representative of two technical replicates.
Extended Data Figure 2 Genetic ablation of Ctr1 decreases BRAFV600E-mediated cell growth and tumorigenesis.
a, Cell growth, as measured by staining with crystal violet, of BRAFV600E-transformed Ctr1+/+ (black circles) or Ctr1−/− (red squares) MEFs (plated in sextuplicate) over a period of three days. Representative of three independent experiments using the same cells. b, c, Representative resected tumours (scale bar, 1 cm) at 20 days after injection (b) and Kaplan–Meier analysis of percentage of mice with tumour volume at least 1.0 cm3 versus time (c) of mice (n = 8) injected with BRAFV600E-transformed Ctr1+/+ (black line) or Ctr1−/− (red line) MEFs. Results were compared using a one-tailed unpaired t-test (a) or a Mantel–Cox test (c). Four asterisks, P < 0.0001.
Extended Data Figure 3 Identification of Cu-binding mutants of MEK1 that decrease ERK1/2 phosphorylation.
a, Immunoblot detection of the amount of HA-tagged wild-type MEK1 and an example of one MEK1 mutant tested (H188A) that bound to a Cu-charged resin. Input serves as a loading control. b, Immunoblot detection of the amount of phosphorylated (P) and/or total (T) ERK1/2 or HA-MEK1 protein in Ctr1+/+ MEFs stably expressing HA-tagged wild-type MEK1 or an example of one MEK1 mutant tested (H188A). c, Summary of whether the indicated MEK1 point mutants did (YES) or did not (NO) show a decrease in binding to the Cu-charged resin or show a decrease in the levels of phosphorylated (P) ERK1/2 when stably expressed in Ctr1+/+ MEFs. Gel images are representative of two technical replicates.
Extended Data Figure 4 Amino acids in MEK1 identified to be oxidized by the MCO reaction followed by MS/MS.
Representative annotated MS/MS fragmentation spectra for five indicated MEK1-derived peptides containing oxidized residues highlighted in red: a, MEK1H87 and MEK1M94; b, MEK1H100; c, MEK1H188; d, MEK1M230; e, MEK1H239. The peak heights are the relative abundances of the corresponding fragmentation ions, with the annotation of the identified matched amino-terminus-containing ions (b ions) in blue and the carboxy-terminus-containing ions (y ions) in red. For clarity, only the major identified peaks are labelled. f, Amino-acid sequence of human MEK1 with the peptides identified by MS/MS underlined (red, trypsin digest; blue, chymotrypsin digest). Amino acids oxidized only in the presence of H2O2 in one to three independent MCO reactions are denoted in red. Boxes enclose amino acids that when mutated to alanine decreased both the binding of MEK1 to a Cu-charged resin and the phosphorylation of cellular ERK2 (from Extended Data Fig. 3c).
Extended Data Figure 5 Alignment of the amino-acid sequences of MEK1, MEK2, MEK5 and MEK1–MEK5.
The amino-acid sequences of human MEK1, MEK2, MEK5 and the MEK1–MEK5 chimaeric protein (without the DD mutation) aligned using Clustal W. Black letters, amino acids; coloured letters, the four amino acids mutated in MEK1CBM to decrease Cu binding (blue, conserved between MEK1, MEK2 and MEK5; red, conserved only between MEK1 and MEK2). Dashes indicate gaps in the alignment.
Extended Data Figure 6 Protein purification and biochemical analysis of wild-type and CBM versions of MEK1.
a, Coomassie blue detection of the amount of wild-type or CBM mutant purified recombinant GST-tagged MEK1 protein in the absence or presence of precision protease for cleavage of GST. b–d, Circular dichroism spectra at increasing wavelengths (b), thermal denaturation monitored at 222 nm at increasing temperature (c), and differential scanning fluorimetry at increasing temperature (d, left) and the average estimated melting temperature (d, right) of purified recombinant MEK1WT (black circles and line) and MEK1CBM (red squares and line). Data are representative of two technical replicates.
Extended Data Figure 7 Tumorigenic growth of NRAS mutation-positive human melanoma cancer cell lines on knockdown of CTR1.
a, b, RT–PCR detection of the amount of endogenous CTR1 and GAPDH mRNA (a) and mean tumour volume (±s.e.m.) versus time (b) of mice (n = 3) injected with the NRAS mutation-positive (NRASQ61L) human melanoma cell lines DM598 and DM792 stably infected with a retrovirus expressing either a scramble (SCRAM) shRNA (black circles) or CTR1 shRNA (red squares). Results were compared using a one-tailed unpaired t-test (b). Four asterisks, P < 0.0001.
Extended Data Figure 8 Detection of Cre-mediated recombination and weight measurements of AdCre-treated Ctr1+/+versus Ctr1flox/floxBP mice.
a, PCR detection of BrafCA/+, Trp53flox/flox and Ctr1flox/flox recombined alleles from matched tail samples (T) and lung tumour cell lines (C) generated from indicated genotypes. Alleles are indicated by arrowheads as follows: black, WT; red, flox; blue, null; orange, BrafCA; green, BrafV600E. b, Box-and-whiskers plot of weight of Ctr1+/+ versus Ctr1flox/flox BP mice (n = 30) one month after intranasal treatment with AdCre. Results were compared using a one-tailed unpaired t-test (b). Four asterisks, P < 0.0001.
Extended Data Figure 9 TTM does not decrease the weight of mice with tumours.
Mean weight (±s.e.m.) over time of mice (n = 4) injected with BRAFV600E-transformed MEFs and treated with vehicle (black circles) or TTM (red squares).
Extended Data Figure 10 Graphical representation of Cu regulation of BRAFV600E-mediated signalling and tumorigenesis.
Inactivation of the signalling pathway is denoted in grey and dashed lines, gain-of-function mutations are denoted in green, and loss-of-function mutations are denoted in red.
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Brady, D., Crowe, M., Turski, M. et al. Copper is required for oncogenic BRAF signalling and tumorigenesis. Nature 509, 492–496 (2014). https://doi.org/10.1038/nature13180
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DOI: https://doi.org/10.1038/nature13180
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