Copper is an essential regulator of the autophagic kinases ULK1/2 to drive lung adenocarcinoma

Abstract

Although the transition metal copper (Cu) is an essential nutrient that is conventionally viewed as a static cofactor within enzyme active sites, a non-traditional role for Cu as a modulator of kinase signalling is emerging. Here, we found that Cu is required for the activity of the autophagic kinases ULK1 and ULK2 (ULK1/2) through a direct Cu–ULK1/2 interaction. Genetic loss of the Cu transporter Ctr1 or mutations in ULK1 that disrupt the binding of Cu reduced ULK1/2-dependent signalling and the formation of autophagosome complexes. Increased levels of intracellular Cu are associated with starvation-induced autophagy and are sufficient to enhance ULK1 kinase activity and, in turn, autophagic flux. The growth and survival of lung tumours driven by KRASG12D is diminished in the absence of Ctr1, is dependent on ULK1 Cu binding and is associated with reduced levels of autophagy and signalling. These findings suggest a molecular basis for exploiting Cu-chelation therapy to prevent autophagy signalling to limit proliferation and improve patient survival in cancer.

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Fig. 1: Cu binds to ULK1 and ULK2 and is required for kinase activity.
Fig. 2: Genetic ablation or enhancement of the intracellular Cu levels modulates autophagic flux.
Fig. 3: Cu is both necessary and sufficient for autophagosome formation.
Fig. 4: Genetic ablation of Ctr1 decreases proliferation to reduce tumorigenesis in a mouse model of KrasG12D-driven lung cancer.
Fig. 5: Genetic ablation of Ctr1 decreases autophagy to reduce tumorigenesis and sensitizes cancer cells to starvation in a mouse model of KrasG12D-driven lung cancer.
Fig. 6: Binding of Cu to ULK1 is required for autophagy signalling and induction.
Fig. 7: Binding of Cu to ULK1 is required for autophagosome complex formation.
Fig. 8: Binding of Cu to ULK1 is required for KRASG12D-driven tumour growth and cancer cell survival in response to starvation.

Data availability

All data generated that support the findings of this study are available from the corresponding author on reasonable request. Unprocessed blots have been provided for Figs. 1b–j, 2a,e, 5g, 6a–f and 8a,b, and Extended Data Figs. 1a, 2d,e,g,i,j,l, 6a–c,e,f and 7d. Source data have been provided for Figs. 2b,d,f,h, 3b,g–l, 4f,g,i, 5b,d,f,j, 6e, 7c–e and 8c,e,g, and Extended Data Figs. 1b–k, 2a–c,f,h,k, 3b,e,f, 4b,c, 5b–d,f, 6d,f,h,i and 7a,c,e–g.

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Acknowledgements

We thank D. J. Thiele (Duke University), C. J. Chang (University of California Berkeley), R. K. Amaravadi (University of Pennsylvania) and S. A. Tooze (The Francis Crick Institute) for reagents, and R. K. Amaravadi, I. A. Asangani, L. Busino, C. V. Dang, J. M. Davis, T. P. Gade, K. E. Hamilton, B. Keith, M. E. Murphy, R. Natesan, A. M. O’Reilly, S. W. Ryeom, M. C. Simon, B. Z. Stanger, J. Tobias, N. A. Tripp, A. T. Weerartna, K. E. Wellen and E. S. Witze for technical support, discussions and/or reading the manuscript. This work was supported by the American Cancer Society Postdoctoral Fellowship (131203 PF 17 147 01 CCG, to J.M.P.), NIH grants GM124749 (to D.C.B.), CA193603 (to D.M.F.), CA222503 (to D.M.F.), ES019851 (to M.C.) and CA243294 (T.T.), Pew Scholars Program in Biomedical Science Award no. 50359 (to D.C.B.), and V Foundation Scholar Award 3C59 8ABS 3424 3BDA (to D.C.B.).

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Authors

Contributions

T.T. and J.M.P. contributed equally to this research. T.T. contributed to the study design, generated cell lines and plasmids, prepared samples for ICP-MS, performed western-blot analysis, immunocomplex kinase assays, crystal-violet growth assays, co-immunoprecipitations, qPCR and in vivo xenograft mouse work, analysed data and prepared figures. J.M.P. generated cell lines, performed flow cytometry, immunofluorescence imaging, live-cell imaging, immunohistochemical analysis, western-blot analysis, crystal-violet growth assays and qPCR, and assisted with in vivo KRASG12D mouse tumour model work. A.A.G. maintained in vivo KRASG12D mouse tumour models and performed in vivo KRASG12D mouse tumour model imaging. M.C. generated in vivo KRASG12D mouse model tumours. D.M.F. contributed to the study design, provided expertise about in vivo mouse tumour models and facilitated data analysis. D.C.B. conceived the project, contributed to the study design, generated cell lines, plasmids and recombinant proteins, performed Cu-binding assays, in vitro kinase assays, immunofluorescence imaging and in vivo xenograft mouse research, analysed data, prepared figures and wrote the manuscript. All of the authors read and provided feedback on manuscript and figures.

Corresponding author

Correspondence to Donita C. Brady.

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Competing interests

D.C.B. holds ownership in Merlon Inc. D.C.B. is an inventor on the patent application US 20150017261 entitled “Methods of treating and preventing cancer by disrupting the binding of copper in the MAP kinase pathway”. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Cu is both necessary and sufficient for ULK1 and ULK2 kinase activity.

ad, Scatter dot plot with bar at mean normalized ΔPhosphorylated (P)-ATG13/Total (T)-ATG13 from Fig. 1d, e, g. n = 3 biologically independent experiments, Fig. 1d, e, g, or n = 4 biologically independent experiments, Fig. 1f. Results were compared using a one-way ANOVA followed by a Tukey’s multi-comparisons test. 0 vs. 50 μM TTM, *, P = 0.0151; 0 vs. 10 μM MRT68921, *, P = 0.0395. e, RT-PCR detection of indicated mRNAs from Ctr1+/+ MEFs or Ctr1-/- MEFs stably expressing CTR1WT (WT) or empty vector (VO). n = 1 biologically independent sample. f, Scatter dot plot of inductively coupled plasma mass spectrometry (ICP-MS) detection with bar at mean Cu (parts per million, ppm) from Ctr1+/+ MEFs ( + /+) or Ctr1-/- MEFs stably expressing WT or VO per sample weight ± s.e.m. n = 3 biologically independent samples. Results were compared using a one-way ANOVA followed by a Tukey’s multi-comparisons test. *, P = 0.05 0.0202; **, P = 0.0059. g, Representative live cell imaging of the Cu probe CF4 or control Cu probe Ctrl-CF4 from Ctr1+/+ MEFs ( + /+) or Ctr1-/- MEFs stably expressing WT or VO. Scale bar, 100 μm. h, Scatter dot plot with bar at mean CF4 or Ctrl-CF4 fluorescence intensity (FI) ± s.e.m. from Ctr1+/+ MEFs ( + /+) or Ctr1-/- MEFs stably expressing WT or VO. n = 90 individual cells. Results were compared using a one-way ANOVA followed by a Tukey’s multi-comparisons test. ****, P < 0.0001. i–k, Scatter dot plot with bar at mean normalized ΔP-ATG13/T-ATG13 from Fig. 1h–j. n = 3 biologically independent experiments. Results were compared using a one-way ANOVA followed by a Tukey’s multi-comparisons test, an unpaired, two-tailed Student’s t-test, or two-way ANOVA followed by a Sidak’s multi-comparisons test. ns. Source data

Extended Data Fig. 2 Copper is both necessary and sufficient for autophagy induction and signaling in a MAPK signaling independent fashion, upstream of ATG5.

a, Scatter dot plot with bar at mean normalized ΔLC3-II/β-ACTIN from Fig. 2a. n = 3 biologically independent experiments. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. ns. b, Scatter dot plot with bar at mean normalized expression of Atp7a mRNA from MEFs n = 1 biologically independent experiment performed in technical triplicate. c, Scatter dot plot with bar at mean normalized ΔLC3-II/β-ACTIN from Fig. 2e. n = 4 biologically independent experiments. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. ns. d, Immunoblot detection of proteins from MEFs. e, Immunoblot detection of proteins from treated MEFs. Quantification: ΔLC3-II/β-ACTIN normalized to fl/fl, empty vector (-), VEH control. f, Scatter dot plot with bar at mean normalized ΔLC3-II/β-ACTIN from Extended Data Fig 2e. n = 3 biologically independent experiments. Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. ns. g, Immunoblot detection of proteins from treated MEFs. Quantification: ΔLC3-II/β-ACTIN normalized to WT, VEH control. h, Scatter dot plot with bar at mean normalized ΔLC3-II/β-ACTIN from Extended Data Fig 2g. n = 6 biologically independent experiments. Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. ns. i, Immunoblot detection of proteins from MEFs. j, Immunoblot detection of proteins from treated MEFs. Quantification: ΔLC3-II/β-ACTIN normalized to WT, VEH control. k, Scatter dot plot with bar at mean normalized ΔLC3-II/β-ACTIN from Extended Data Fig 2j. n = 5 biologically independent experiments. Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. *, P = 0.0071; **, P = 0.0434; ***, P = 0.0026; ****, P = 0.0021. l, Immunoblot detection of proteins from treated MEFs. Western blot images are representative of at least three biological replicates. Source data

Extended Data Fig. 3 Cu but not Fe is required for autophagosome formation and is associated with fluctuations in the Cu labile pool.

a, Representative live cell imaging of the Cu probe Ctrl-CF4 every ten minutes for 60 min from MEFs treated with vehicle (VEH) or amino acid deprivation (-AA). Scale bar, 100 μm. b, Mean Ctrl-CF4 fluorescence intensity (FI) ± s.e.m. versus time (minutes, min) from MEFs treated with VEH or -AA normalized to t = 0, five minutes. n = 30 individual cells. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. c, Schematic of immunofluorescence-based approach to access autophagosome formation. d, Schematic of flow cytometry-based approach to access autophagosome number. e, f, Scatter dot plot with bar at mean GFP-LC3 fluorescent intensity ± s.e.m. analyzed by flow cytometry from MEFs stably expressing EGFP-LC3B treated VEH or increasing concentrations of Cu chelator TTM (e) or Fe chelator DFO (f). n = 9 biologically independent samples. Results were compared using a one-way ANOVA followed by a Dunnett’s multi-comparisons test. *, P = 0.0148; ****, P < 0.0001. Source data

Extended Data Fig. 4 Genetic ablation of Ctr1 reduces KrasG12D-driven lung tumorigenesis.

a, Normalized representative images of in vivo luminescence of KrasG12D/+;Trp53flox/flox;Rosa26::LSL-Luc (KPLuc) mice introduced with either sgRNA against Glb1 or Ctr1 at week 16 endpoint. b, c, Scatter dot plot with bar at mean ΔLuminescence units from week 10 to week 16 from KPLuc mice introduced with either sgRNA against Glb1 or Ctr1. n = 6 biologically independent animals. Results were compared using an unpaired, one-tailed Student’s t-test. P = 0.0818. Source data

Extended Data Fig. 5 Genetic ablation of Ctr1 decreases MAPK signaling to reduce KrasG12D-driven lung tumorigenesis, while survival in response starvation is independent of MAPK signaling.

a, Representative 40x images of immunohistochemical detection of phosphorylated (P)-ERK1/2 of lungs from KPLuc mice expressing sgRNA against Glb1 or Ctr1. (40x scale bar: 50 µm). b, Scatter dot plot with bar at mean ± s.e.m. % P-ERK1/2 positive staining per tumor (Glb1 and Ctr1) from KPLuc mice expressing sgRNA against Glb1 or Ctr1. n = 66 images. Results were compared using an unpaired, two-tailed Student’s t-test. ***, P = 0.0002. c, Scatter dot plot with bar at mean normalized quantitative PCR (qPCR) expression of Ctr1 mRNA from KP lung adenocarcinoma cell lines #54 (KP #54) and #2474 (KP #2474) stably expressing sgRNA against Rosa (-) or Ctr1 (#1 or #2). n = 1 biologically independent experiment performed in technical triplicate. d, Scatter dot plot with bar at mean normalized ΔLC3-II/β-ACTIN from Fig. 5g. n = 3 independent experiments. Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. ns. e, Representative crystal violet images of KP #54 and KP #2474 cells stably expressing sgRNA against Rosa, Atg5, or Ctr1 (#1 or #2) and ERK2GOF from days 3, 4, and 5 of recovery. f, Scatter dot plot with bar at mean absorbance of extracted crystal violet at 590 nM ± s.e.m. of KP #54 and KP #2474 cells stably expressing sgRNA against Rosa, Atg5, or Ctr1 (#1 or #2) and gain-of-function (GOF) HA-ERK2 (ERK2GOF) from days 3, 4, and 5 of recovery normalized to Rosa, day 3 control. KP #54, n represents number of biologically independent samples; Day 3 n = 9, Day 4 n = 9, Day 5, Rosa n = 8, Atg5 n = 9, #1 n = 9, #2 n = 9. *, P = 0.0140; ***, P = 0.0003. KP #2474, n represents number of biologically independent samples; Day 3 n = 9, Day 4 n = 9, Day 5, Rosa n = 9, Atg5 n = 9, #1 n = 8, #2 n = 8. *, P = 0.0011; **, P = 0.0158; ***, P = 0.0462. Source data

Extended Data Fig. 6 Binding of Cu to ULK1 is required for kinase activity but not substrate association or phosphorylation.

a, Immunoblot detection of phosphorylated (P) ATG13, total (T)- ATG13, P-ULK1 (S555), P-ULK1 (S757), T-ULK1, or β-ACTIN from Ulk1/2-/- MEFs stably expressing HA-ULK1WT (WT) or HA-ULK1CBM (CBM) treated with vehicle (VEH) or amino acid deprivation (-AA). b, Immunoblot detection of T-ULK1 or β-ACTIN from cell lysates treated with or without calf alkaline phosphatase (CIP) from Ulk1/2-/- MEFs stably expressing empty vector (VO), HA-ULK1WT (WT), or HA-ULK1CBM (CBM). c, Immunoblot detection of T-ATG13, T-ATG101, T-FIP200, T-ULK1, or β-ACTIN from immunoprecipitated (IP)-ULK1 or whole cell extracts (WCE) from Ulk1/2-/- MEFs stably expressing HA-ULK1WT (WT) or HA-ULK1CBM (CBM) treated with VEH or -AA. d, Scatter dot plot with bar at mean normalized quantitative PCR (qPCR) expression of Ulk1 or Ulk2 mRNA from MEFs stably expressing sgRNA against Rosa or Ulk1 and Ulk2. n = 1 biologically independent experiment performed in technical triplicate. e, Immunoblot detection of T-ULK1, T-ULK2, or β-ACTIN from MEFs stably expressing sgRNA against Rosa (-) or Ulk1 and Ulk2 ( + ). f, Immunoblot detection of LC3-I, LC3-II, or β-ACTIN from MEFs stably expressing sgRNA against Rosa (-) or Ulk1 and Ulk2 ( + ) treated with -AA and bafilomycin (BAF) for 1 h (hr), 2 hr, and 3 hr. gi, Scatter dot plot with bar at mean normalized ΔLC3-II/β-ACTIN or ΔP-ATG13/T-ATG13 from Fig. 6d, f. n = 3 independent experiments. Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. g, ns; h, ****, P < 0.0001. i, *, P = 0.0365; ****, P < 0.0001. Western blot images are representative of at least three biological replicates. Source data

Extended Data Fig. 7 Binding of Cu to ULK1 is required for tumorigenesis by oncogenic KRASG12D.

a, Mean tumor volume (mm3) ± s.e.m. versus time (days) in mice injected with Ulk1-/- MEFs stably expressing either HA-ULK1WT or HA-ULK1CBM and transformed with KRASG12D. n = 4 biologically independent animals. Results were compared using a paired, one-tailed Student’s t-test. **, P = 0.0095. b, Representative dissected tumors from mice injected with Ulk1-/- MEFs stably expressing either HA-ULK1WT (WT) or HA-ULK1CBM (CBM) and transformed with KRASG12D. Scale bar, 100 µm. c, Scatter dot plot with bar at mean tumor weight (g) ± s.e.m. of tumors at endpoint from Ulk1-/- MEFs stably expressing either WT or CBM and transformed with KRASG12D. n = 4 biologically independent samples. Results were compared using an unpaired, one-tailed Student’s t-test. *, P = 0.0325. n = 4. d, Immunoblot detection of T-ULK1, T-ULK2, or β-ACTIN from KrasG12D/+;Trp53flox/flox (KP) lung adenocarcinoma cell line #2474 (KP #2474) stably expressing sgRNA against Rosa (-) or Ulk1 and Ulk2 ( + ). e, f, Scatter dot plot with bar at mean normalized ΔLC3-II/β-ACTIN or ΔP-ATG13/T-ATG13 from Fig. 8a, b. n = 3 independent experiments. Results were compared using a one-way ANOVA or a two-way ANOVA followed by a Tukey’s multi-comparisons test. e, **, P = 0.0013; ***, P = 0.0016; ****, P < 0.0001; f, *, P = 0.0113; **, P = 0.0063; ***, P = 0.0113. g, Scatter dot plot with bar at mean normalized quantitative PCR (qPCR) expression of Ctr1 mRNA from KP #2474 cells stably expressing sgRNA against Rosa, Ctr1 #2, or Ulk1, Ulk2, and Ctr1 #2. n = 1 biologically independent experiment performed in technical triplicate. Source data

Supplementary information

Supplementary Information

Supplementary Fig. 1: an example of the gating strategy for the FACS experiments.

Reporting Summary

Supplementary Video 1

Representative live-cell imaging of the Cu probe CF4 every 10 min for 60 min from MEFs treated with vehicle.

Supplementary Video 2

Representative live-cell imaging of the Cu probe CF4 every 10 min for 60 min from MEFs treated with amino acid deprivation.

Supplementary Video 3

Representative live-cell imaging of the Cu probe Ctrl-CF4 every 10 min for 60 min from MEFs treated with vehicle.

Supplementary Video 4

Representative live-cell imaging of the Cu probe Ctrl-CF4 every 10 min for 60 min from MEFs treated with amino acid deprivation.

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Tsang, T., Posimo, J.M., Gudiel, A.A. et al. Copper is an essential regulator of the autophagic kinases ULK1/2 to drive lung adenocarcinoma. Nat Cell Biol 22, 412–424 (2020). https://doi.org/10.1038/s41556-020-0481-4

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