Abstract

For cancer cells to survive during extracellular matrix (ECM) detachment, they must inhibit anoikis and rectify metabolic deficiencies that cause non-apoptotic cell death. Previous studies in ECM-detached cells have linked non-apoptotic cell death to reactive oxygen species (ROS) generation, although the mechanistic underpinnings of this link remain poorly defined. Here, we uncover a role for receptor-interacting protein kinase 1 (RIPK1) in the modulation of ROS and cell viability during ECM detachment. We find that RIPK1 activation during ECM detachment results in mitophagy induction through a mechanism dependent on the mitochondrial phosphatase PGAM5. As a consequence of mitophagy, ECM-detached cells experience diminished NADPH production in the mitochondria, and the subsequent elevation in ROS levels leads to non-apoptotic death. Furthermore, we find that antagonizing RIPK1/PGAM5 enhances tumour formation in vivo. Thus, RIPK1-mediated induction of mitophagy may be an efficacious target for therapeutics aimed at eliminating ECM-detached cancer cells.

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Acknowledgements

We thank Veronica Schafer, all current and past Schafer lab members, James Clancy, Crislyn D’Souza-Schorey, H. Clay Conner, Athanasia Panopoulos, Ian Guldner, Siyuan Zhang, Rebecca Wingert and Paul Huber for helpful comments, experimental assistance and valuable discussion. We thank Jeffrey Hawk, Janet Hawk and Veronica Wende for helpful comments and support. We also thank Leta Nutt (St. Jude Children’s Research Hospital) for the Bax(−/−)/Bak(−/−) MEFs, Mary Ann McDowell (Notre Dame) for assistance with immunofluorescence, Kevin T. Vaughan (Notre Dame) for help with soft agar and William Archer (Notre Dame) for guidance with immunofluorescence. This work was supported by a Lee National Denim Day Research Scholar Grant (RSG-14-145-01-CSM) from the American Cancer Society (to Z.T.S.), a research grant from the Phi Beta Psi National Project (to Z.T.S.), a National Science Foundation Graduate Research Fellowship grant DGE 1313583 (to M.A.H.), the Coleman Foundation (Chicago, IL), and funds from Ron and Rosemarie Malanga.

Author information

Affiliations

  1. Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, USA

    • Mark A. Hawk
    • , Cassandra L. Gorsuch
    • , Patrick Fagan
    • , Joshua A. Mason
    • , Kelsey J. Weigel
    • , Matyas Abel Tsegaye
    • , Luqun Shen
    • , Sydney Shuff
    • , Junjun Zuo
    • , Stephan Hu
    • , Sarah Chapman
    • , W. Matthew Leevy
    •  & Zachary T. Schafer
  2. Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    • Chan Lee
    • , Sung Eun Kim
    • , Jens C. Hamann
    •  & Michael Overholtzer
  3. BCMB Allied Program, Weill Cornell Medical College, New York, NY, USA

    • Chan Lee
    • , Sung Eun Kim
    •  & Michael Overholtzer
  4. Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    • Jens C. Hamann
    •  & Michael Overholtzer
  5. Department of Molecular and Cellular Endocrinology, Beckman Research Institute at City of Hope, Duarte, CA, USA

    • Lei Jiang
  6. Children’s Medical Center Research Institute, UT Southwestern Medical Center, Dallas, TX, USA

    • Ralph J. DeBerardinis
  7. Department of Pediatrics, UT Southwestern Medical Center, Dallas, TX, USA

    • Ralph J. DeBerardinis
  8. McDermott Center for Human Growth and Development, UT Southwestern Medical Center, Dallas, TX, USA

    • Ralph J. DeBerardinis

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Contributions

M.A.H., C.L.G., P.F., J.A.M., K.J.W., M.A.T., L.S., S.S., J.Z. and S.H. conducted experiments, analysed data and interpreted results. C.L., S.E.K., J.C.H. and M.O. assisted with the imaging and quantitation of necrotic cells and provided the ATG5 knockout MCF-10A cells. L.J. and R.J.D. provided H460 cells with IDH1 or IDH2 deficiencies and assisted with analysis and interpretation of related experiments. S.C. and W.M.L. carried out the in vivo studies in immunocompromised mice. M.A.H. and Z.T.S. wrote the manuscript with feedback from all other authors. Z.T.S. was responsible for conception/design of the project and overall study supervision.

Competing interests

R.J.D. is an advisor for Agios Pharmaceuticals. All other authors declare no conflicts of interest.

Corresponding author

Correspondence to Zachary T. Schafer.

Integrated supplementary information

  1. Supplementary Figure 1 NF-κB promotes CYLD transcription during ECM-detachment and overexpression of RIPK1 lowers cell viability.

    (a) MCF-10A EV cells were grown in either Att or Det conditions for 24h. Lysates were immunoblotted as noted. (b) Human mammary epithelial cells (HMEC) were grown in either Att or Det conditions for 24h. Lysates were immunoblotted as noted. (c) 10A-Bcl2 cells were grown in either Att or Det conditions for 24h. Lysates were immunoblotted as noted. (d) 10A-Bcl2 cells were grown in Att or Det for 24h. Relative expression of CYLD was measured by qRT-PCR. n=3 independent biological samples. (e, f) 10A-Bcl2 cells were grown in Att or Det for 24h treated with either DMSO or Bay-117082 (10μM). Relative expression of CYLD was measured by qRT-PCR. n=3 independent biological samples (e). Lysates were immunoblotted as noted (f). (g) HMEC cells were grown in Att or Det conditions treated with DMSO or Nec1 (10μM) for 24h. Cell viability was measured using AlamarBlue. n=3 independent biological samples. (h) 10A-Bcl2 cells transfected with empty vector (EV) or RIPK1 wildtype (RIPK1 WT) were grown in Att for 24h. Lysates were immunoblotted as noted. (i) Cell viability was measured using AlamarBlue following transfection of Att 10A-Bcl2 cells after being treated with either DMSO or Nec1 (10μM) for 24h. n=3 independent biological samples. (j) Left panel, Bax/Bak null (Bax(-/-)/Bak(-/-)) MEFs transfected with empty vector (EV), RIPK1 wildtype (RIPK1 WT), or RIPK1 kinase mutant (RIPK1 K45A) were grown in Att for 24h. Lysates were immunoblotted as noted. (k) Cell viability was measured using AlamarBlue following transfection after being grown in Att for 24h. n=3 independent biological samples. (l) BT474, T47D, and MCF7 cells were grown in either Att or Det conditions for 24h. Lysates were immunoblotted as noted. (m) HeLa, HCT116, and H460 cells were grown in either Att or Det conditions for 24h. Lysates were immunoblotted as noted. All results are presented as means +/- SEM and statistical significance was calculated using a Student’s two tailed t-test. p<0.05 is statistically significant. All western blotting experiments were independently repeated a minimum of three times with similar results. Unprocessed original scans of blots are available in Supplementary Fig. 9.

  2. Supplementary Figure 2 Caspase activation is not impacted by Nec1-mediated RIPK1 inhibition.

    (a-f) BT474 (a), T47D (b), MCF7 (c), HeLa (d), HCT116 (e), and H460 (f) cells were grown in Att and Det after being treated with DMSO or Nec1 (10μM) for 48h. Caspase-3/7 activation was measured 48h after plating in Att and Det using the CaspaseGlo 3/7 assay. Results are presented as means +/- SEM and statistical significance was calculated using a Student’s two tailed t-test. Caspase activation is represented as a ratio of Det value over Att value. n=3 independent biological samples for each cell line. N.S., not significant. (g) 10A-Bcl2 cells were grown in soft agar and imaged each day for three days. Cells were scored as viable or necrotic as indicated. Results are presented as percentage of necrotic cells +/- SD. n=12 independent biological samples. (h) 10A-Bcl2 cells were grown in soft agar and imaged each day for three days. Cells were scored as viable or entotic as indicated. Results are presented as percentage of entotic cells +/- SD. n=12 independent biological replicates.

  3. Supplementary Figure 3 RIPK3 nor MLKL are required for ECM-detachment induced RIPK1 signalling.

    (a, b) MCF-10A pLKO (a,b), shRIPK3 (a) and shMLKL (b) cells were grown in Att and Det for 48h. Caspase-3/7 activation was measured 48h after plating in Att and Det using the CaspaseGlo 3/7 assay. Caspase activation is represented as a ratio of Det value over Att value. n=3 independent biological samples. (c) Immunoblotting against RIPK3 and β actin was used to confirm the RIPK3 knockdown (shRIPK3) in H460 cells grown in Det for 24h. Experiments were performed a minimum of three independent times with similar results. (d-f) Cell viability (d), caspase activation (e), and anchorage-independent growth in soft agar (f) was measured in Att and Det H460 pLKO and shRIPK3 cells at 24h. Viability was measured using AlamarBlue, caspase activation using CaspaseGlo 3/7 assay, and after 7 days, images of soft agar growth taken following INT-violet staining. Colony count was determined using ImageJ. n=6 independent biological replicates for soft agar and n=3 independent biological samples for viability and caspase measurements. (g) Immunoblotting against MLKL and β actin was used to confirm the MLKL knockdown (shMLKL) in H460 cells grown in Det for 24h. Experiments were performed a minimum of three independent times with similar results. (h-j) Cell viability (h), caspase activation (i), and anchorage-independent growth in soft agar (j) was measured in Att and Det H460 pLKO and shMLKL cells at 24h. Viability was measured using AlamarBlue, caspase activation using CaspaseGlo 3/7 assay, and after 7 days, images of soft agar growth taken following INT-violet staining. Colony count was determined using ImageJ. n=6 biological independent biological replicates for soft agar and n=3 independent biological samples for viability and caspase measurements. All results are presented as means +/- SEM and statistical significance was calculated using a Student’s two tailed t-test. N.S., not significant. Unprocessed original scans of blots are available in Supplementary Fig. 9

  4. Supplementary Figure 4 PGAM5 is downstream of RIPK1-mediated loss of viability.

    (a) Immunoblotting against PGAM5 and β actin was used to confirm the PGAM5 knockdown (shPGAM5) in MCF-10A cells grown in Att for 24h. Experiments were performed a minimum of three independent times with similar results. (b) MCF-10A pLKO and shPGAM5 cells transfected with empty vector (EV) or RIPK1 wildtype (RIPK1 WT) were grown in Att for 24h. Lysates were then immunoblotted for total RIPK1 and β actin. Experiments were performed a minimum of three independent times with similar results. (c, d) Cell viability was measured using AlamarBlue following transfection of MCF-10A pLKO (c) and shPGAM5 (d) cells after being grown in Att for 24h. n=3 independent biological samples. (e) Immunoblotting against PGAM5 and β actin was used to confirm the PGAM5 knockdown (shPGAM5) in H460 cells grown in Att for 24h. Experiments were performed a minimum of three independent times with similar results. (f) H460 pLKO and shPGAM5 cells transfected with empty vector (EV) or RIPK1 wildtype (RIPK1 WT) were grown in Att for 24h. Lysates were immunoblotted for Myc tag, and β actin to confirm the overexpression of Myc-tagged-RIPK1. Experiments were performed a minimum of three independent times with similar results. (g, h) Cell viability was measured using AlamarBlue following transfection H460 pLKO (g) and shPGAM5 (h) cells after being grown in Att for 24h. n=3 independent biological samples. All results are presented as means +/- SEM and statistical significance was calculated using a Student’s two tailed t-test. p<0.05 is statistically significant. N.S., not significant. Unprocessed original scans of blots are available in Supplementary Fig. 9.

  5. Supplementary Figure 5 Inhibition of RIPK1 or PGAM5 blocks ECM-detachment-induced mitophagy.

    (a) H460 cells were grown in Att or Det for 24h treated with DMSO, Nec1 (10μM), BAFA (10nM) or CCCP (10μM). Cells were then cytospun onto slides, fixed, and stained with Mito-Tracker Red (200nM) (red) and DAPI (blue). Representative images of each condition are shown (40x). (b) Relative fluorescence measurements for each condition in (a) were determined using Fiji. For fluorescence calculations, n = 5 images. (c) H460 pLKO, shRIPK1, shPGAM5, and shPINK1 cells grown in Att or Det for 24h. Cells were then cytospun onto slides, fixed, and stained with Mito-Tracker Red (200nM) (red) and DAPI (blue). Representative images of each condition are shown (40x). (d) Relative fluorescence measurements for each condition in (c) were determined using Fiji. For fluorescence calculations, n = 5 images. (e) 10A-Bcl2 cells were grown in Att or Det for 24h treated with DMSO, Nec1 (10μM), or Nec1 (10μM) and CCCP (10μM). Cell viability was measured using AlamarBlue. Viability measurements are normalized to Att 10A-Bcl2 DMSO. n=3 independent biological samples. All results are presented as means +/- SEM and statistical significance was calculated using a Student’s two tailed t-test. p<0.05 is statistically significant. Scale bars, 100 μm.

  6. Supplementary Figure 6 Deficiency of ATG5 halts RIPK1-dependent mitophagy during ECM-detachment and mitochondrial ROS generation during ECM-detachment is a consequence of RIPK1-mediated mitophagy.

    (a) Immunoblotting against ATG5-ATG12 conjugate and GAPDH was used to confirm the ATG5 knockout clone in MCF-10A cells using CRISPR/Cas9. Experiments were performed a minimum of three independent times with similar results. (b) MCF-10A EV, ATG5KO, and HA-ATG5-R/E cells were grown in Att or Det for 24h treated with DMSO or Nec1 (10μM). Cell viability was measured using AlamarBlue. n=3 independent biological samples. (c) MCF-10A EV, ATG5KO, and HA-ATG5-R/E cells were grown in Att or Det for 24h treated with DMSO or Nec1 (10μM). Lysates were immunoblotted for Tom20 and β actin. Experiments were performed a minimum of three independent times with similar results. (d) 10A-Bcl2 cells were grown in Att or Det for 24h treated with DMSO, Nec1 (10μM), BAFA (10nM), MitoTEMPO (10μM) or CCCP (10μM). Cells were then cytospun onto slides, fixed, and stained with MitoSOX Red (5μM) (red) and DAPI (blue). Representative images of each condition are shown (40x). (e) Relative fluorescence measurements for each condition in (d) were determined using Fiji. For fluorescence calculations, n = 5 images. (f) H460 cells were grown in Att or Det for 24h treated with DMSO, Nec1 (10μM), BAFA (10nM), MitoTEMPO (10μM) or CCCP (10μM). Cells were then cytospun onto slides, fixed, and stained with MitoSOX Red (5μM) (red) and DAPI (blue). Representative images of each condition are shown (40x). (g) Relative fluorescence measurements for each condition in (f) were determined using Fiji. For fluorescence calculations, n = 5 images. All results are presented as means +/- SEM and statistical significance was calculated using a Student’s two tailed t-test. p<0.05 is statistically significant. N.S., not significant. Scale bars, 100 μm. Unprocessed original scans of blots are available in Supplementary Fig. 9.

  7. Supplementary Figure 7 RIPK1 inhibition, but not ROS neutralization, rescues the loss of mitochondria during ECM-detachment as a consequence of IDH2.

    (a) H460 EV cells were grown in Att or Det for 24h treated with DMSO, Nec1 (10μM), MitoTEMPO (10μM), or M. Malate (2.5mM). Cells were then cytospun onto slides, fixed, and stained with Mito-Tracker Red (200nM) (red) and DAPI (blue). Representative images of each condition are shown (40x). Experiments were performed a minimum of three independent times with similar results. (b) Relative fluorescence measurements for each condition in (a) were determined using Fiji. For fluorescence calculations, n = 5 images. (c) H460 IDH1KO cells were grown in Att or Det for 24h treated with DMSO, Nec1 (10μM), MitoTEMPO (10μM), or M. Malate (2.5mM). Cells were then cytospun onto slides, fixed, and stained with Mito-Tracker Red (200nM) (red) and DAPI (blue). Representative images of each condition are shown (40x). Experiments were performed a minimum of three independent times with similar results. (d) Relative fluorescence measurements for each condition in (c) were determined using Fiji. For fluorescence calculations, n = 5 images. (e) H460 IDH2KO cells were grown in Att or Det for 24h treated with DMSO, Nec1 (10μM), MitoTEMPO (10μM), or M. Malate (2.5mM). Cells were then cytospun onto slides, fixed, and stained with Mito-Tracker Red (200nM) (red) and DAPI (blue). Representative images of each condition are shown (40x). Experiments were performed a minimum of three independent times with similar results. (f) Relative fluorescence measurements for each condition in (e) were determined using Fiji. For fluorescence calculations, n = 5 images. All results are presented as means +/- SEM and statistical significance was calculated using a Student’s two tailed t-test. p<0.05 is statistically significant. N.S., not significant. Scale bars, 100 μm.

  8. Supplementary Figure 8 IDH2 activity is required for cell survival when RIPK1 is inhibited.

    (a-c) H460 EV (a), H460 IDH1KO (b), or H460 IDH2KO (c) cells were grown in Att or Det for 24h treated with DMSO, Nec1 (10μM), MitoTEMPO (10μM), or M. Malate (2.5mM). Cell viability was measured using AlamarBlue. n=3 independent biological samples. (d-f) H460 EV (d), H460 IDH1KO (e), or H460 IDH2KO (f) cells were grown in Att or Det for 24h treated with DMSO, Nec1 (10μM), or M. Malate (2.5mM). The ratio of NADPH/NADP+ was measured using NADP/NADPH-Glo assay. n=3 independent biological samples. (g) H460 EV (g), H460 IDH1KO (h), or H460 IDH2KO (i) cells were grown in Att or Det for 24h treated with H2O or H2O2. Cells were then cytospun onto slides, fixed, and stained with CellROX Green (5 μM) (green) and DAPI (blue). Representative images of each condition are shown (40x). n=3 independent biological samples. All results are presented as means +/- SEM and statistical significance was calculated using a Student’s two tailed t-test. p<0.05 is statistically significant. N.S., not significant. Scale bars, 20 μm.

  9. Supplementary Figure 9

    Unprocessed images of all gels and blots

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–9 and Supplementary Table legends.

  2. Life Sciences Reporting Summary

  3. Supplementary Table 1

    Statistics source data.

  4. Supplementary Table 2

    Primer sequences.

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DOI

https://doi.org/10.1038/s41556-018-0034-2

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