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Limiting mitochondrial plasticity by targeting DRP1 induces metabolic reprogramming and reduces breast cancer brain metastases

Nature Cancer volume 4, pages 893–907 (2023)Cite this article

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Abstract

Disseminated tumor cells with metabolic flexibility to utilize available nutrients in distal organs persist, but the precise mechanisms that facilitate metabolic adaptations remain unclear. Here we show fragmented mitochondrial puncta in latent brain metastatic (Lat) cells enable fatty acid oxidation (FAO) to sustain cellular bioenergetics and maintain redox homeostasis. Depleting the enriched dynamin-related protein 1 (DRP1) and limiting mitochondrial plasticity in Lat cells results in increased lipid droplet accumulation, impaired FAO and attenuated metastasis. Likewise, pharmacological inhibition of DRP1 using a small-molecule brain-permeable inhibitor attenuated metastatic burden in preclinical models. In agreement with these findings, increased phospho-DRP1 expression was observed in metachronous brain metastasis compared with patient-matched primary tumors. Overall, our findings reveal the pivotal role of mitochondrial plasticity in supporting the survival of Lat cells and highlight the therapeutic potential of targeting cellular plasticity programs in combination with tumor-specific alterations to prevent metastatic recurrences.

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Fig. 1: Latent cells uptake FAs secreted by reactive astrocytes.
Fig. 2: Lat cells oxidize internalized FAs and maintain redox homeostasis.
Fig. 3: DRP1-driven mitochondrial dynamics enable FAO and redox homeostasis.
Fig. 4: DRP1-driven mitochondrial plasticity promotes metastatic latency.
Fig. 5: Phospho-DRP1 is elevated in human metachronous brain metastases.
Fig. 6: Genetic depletion or pharmacologic inhibition of DRP1 attenuates brain metastasis.
Fig. 7: Schematic presentation highlighting the role of DRP1-driven mitochondrial plasticity and metabolic reprogramming in HER2+ breast cancer brain metastasis.

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Data availability

The RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus under the accession number GSE180098.The mass spectrometry data have been deposited in Mass Spectrometry Interactive Virtual Environment (MassIVE) with accession number MSV000091574. Source data are provided with this paper. All other data supporting findings of this study can be available from the corresponding author on reasonable request.

Code availability

All of the analyses in our study were conducted using standard workflows and open source software as detailed in Methods, without any custom code being used or developed.

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Acknowledgements

This work was supported by grants from CPRIT training grant (RP210041) to P.K.P., NSF grant (2019281049) to M.M.P., NCI grant (R35CA22044901) to R.J.D., Cancer Center Support Grant (P30 CA142543) and CPRIT (RR170003), ACS (RSG-20-47-01-CSM), METAvivor (GAA202106-0027), Susan G. Komen Career Catalyst Grant (CCR22902470), SCCC Cancer & Obesity Translational Pilot Program Grant, and Breast Cancer–Bone initiative from Charles Y.C. Pak Foundation grants to S.M. We acknowledge Y. Wang for the initial help with astrocytes isolation. We thank K. Luby-Phelps, R. Jackson, P. Doss and A. Lombard from the Electron Microscopy Core Facility and M. Mettlen and A. Bugde from the Quantitative Light Microscopy Core, UT Southwestern Medical Center. We gratefully acknowledge the assistance of preclinical radiation core facility, supported by CPRIT; RP180770 for bioluminescence imaging. Illustrations created with BioRender.com. We acknowledge the UTSW Proteomics Core for assistance with post-translational modification analysis. We are grateful to the patients whose information and samples were vital for this study.

Author information

Authors and Affiliations

  1. Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Pravat Kumar Parida, Mauricio Marquez-Palencia, Suvranil Ghosh, Kangsan Kim, Vidhya Nair, Yan Peng, Cheryl Lewis & Srinivas Malladi

  2. Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Pravat Kumar Parida, Mauricio Marquez-Palencia, Suvranil Ghosh, Kangsan Kim, Vidhya Nair, Yan Peng, Cheryl Lewis, Carlos L. Arteaga, Ariella B. Hanker, Ralph J. DeBerardinis & Srinivas Malladi

  3. Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Nitin Khandelwal

  4. Department of Biomedicine, University of Bergen, Bergen, Norway

    Xiao-Zheng Liu & Nils Halberg

  5. Children’s Research Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Hieu S. Vu, Lauren G. Zacharias & Ralph J. DeBerardinis

  6. Breast Cancer Research Program, Vanderbilt University Medical Center, Nashville, TN, USA

    Paula I. Gonzalez-Ericsson

  7. Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA

    Melinda E. Sanders & Bret C. Mobley

  8. Center for Human Nutrition and Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Jeffrey G. McDonald & Gonçalo Vale

  9. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Andrew Lemoff

  10. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Ariella B. Hanker

  11. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Ralph J. DeBerardinis

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  1. Pravat Kumar Parida
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Contributions

P.K.P. and S.M. conceptualized the project. P.K.P. performed most of the experiments. M.M.P., S.G. and K.K. helped P.K.P. perform in vivo animal experiments and in vitro experiments. N.K. assisted in astrocyte-related work, X.L. and N.H. helped with the FAO assay. R.J.D, H.V., L.G.Z. and V.N. helped in metabolite tracing experiments, A.L. performed DRP1 post-translational modification analysis. G.V. and J.G.M. assisted in FA profiling. P.I.G.-E., M.E.S., B.C.M., A.B.H. and C.L.A. helped in obtaining patient sample resources, Y.P. and C.L. assisted in p-DRP1 IHC staining and analysis in patient samples. Funding and resources are acquired by S.M. The original draft was written by P.K.P. and S.M.; P.K.P., G.V., C.L., A.L., C.L.A., A.B.H., R.J.D. and S.M. reviewed and edited the paper.

Corresponding author

Correspondence to Srinivas Malladi.

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Competing interests

A.B.H. has received research grant support from Takeda and travel support from Puma Biotechnology. C.L.A. receives or has received research grant support from Pfizer, Lilly and Takeda; holds stock options in Provista; and serves or has served in an advisory role to Novartis, Lilly, TAIHO Oncology, Daiichi Sankyo, Merck, AstraZeneca, OrigiMed, Immunomedics and Susan G. Komen Foundation. R.J.D. is a founder of Atavistik Biosciences and an advisor for Agios Pharmaceuticals, Nirogy Therapeutics and Vida Ventures. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Latent cells uptake fatty acids secreted by reactive astrocytes.

a. Showing relative difference in steady-state carnitine-conjugated fatty acids between Pa and Lat cells. n = 4, each group. Box and whiskers plot showing minima to maxima with all points. b. Heatmap showing differential expression of gene sets related to fatty acid metabolism in HCC1954 Pa and Lat cells. c. Western blots showing expression of fatty acid synthesis related enzymes FASN, ACC1 and p-ACC1S79 in HCC1954 Pa and Lat cells. d. 13C6-glucose tracing data showing the distribution of 13C-labeled even isotopologues of palmitate. n = 3, each group. e. Time-lapse confocal images showing BODIPY-558/568-C12 (Orange) transfer from astrocytes to cancer cells (green) in co-culture setting. f. Immunofluorescence images showing accumulation of LDs (red) in SKBR3 Pa and Lat cells (green) cultured with BODIPY-558/568-C12 labeled reactive astrocytes (Gray, GFAP staining). g. Bar graph showing survival of HCC1954 Pa and Lat cells cultured with astrocytes media. Percentage live/dead cells were quantified at 24 and 48 hours, n = 6, each group. h-j. Nile Red staining IF images showing lipid droplets (red) in HCC1954 and SKBR3 Pa and Lat cells treated with lauric acid, palmitic acid, and oleic acid respectively. Briefly, cells were incubated for 24 hours in R3F media and then treated with 100 µM of indicated FAs for 24 hours. Cells were fixed and stained with Nile red (2 µg/ml) for 10 min. k. IncuCyte3 time-lapse images (0,24 and 48 hours) showing lipid uptake and cell death in HCC1954 cells cultured with 2 μM of BODIPY-558/568-C12. In a, d, and g, ‘n’ represents biologically independent samples and data presented as mean + /-SEM. P value in a, d, and g were calculated by two-tailed Unpaired t-test. The experiments shown in c, e, f, and h-k were repeated independently at least two or three times with similar results.

Source data

Extended Data Fig. 2 Fragmented mitochondria puncta and LDs enriched in Latent cells.

a. IncuCyte3 data showing time dependent lipid uptake in HCC1954 Pa and Lat cells in BODIPY-558/568-C12 treated condition. Data were exported as Mean and SEM from n = 25 different fields. P value was calculated by two-tailed Paired t-test. b. Bar graph showing survival of HCC1954 Pa and Lat cells cultured with BODIPY-558/568-C12. Percentage live/dead cells were quantified at 24 and 48 hours, n = 6, each group. c. Transmission electron microscope (TEM) images showing LDs and mitochondria in SKBR3 Pa and Lat cells. SKBR3 Pa and Lat cells were cultured in MatTek dishes for 24 hours with R3F media followed by treatment of sodium palmitate(100 µM) for 24 hours before processed for TEM. d and e. IF images of HCC1954 Pa and Lat cells showing LDs (red) and mitochondria (green). Briefly, cells were treated with 100 µM lauric acid or palmitic acid for 24 hours. Cells were fixed and stained with Nile red followed by anti-TOMM20 antibody labeling was performed to visualize LDs and mitochondria respectively. f. Bar graph showing LDs, in HCC1954 Pa and Lat cells after lauric acid treatment, n = 6, each group. g. Percentage distribution of tubular, intermediate and punctate mitochondrial morphology in HCC1954 Pa and Lat cells treated with lauric acid (n = 4, each group). h. Quantification of LDs in palmitate treated HCC1954 Pa and Lat cells. n = 5, each group. i. Classification of cells based on percentage distribution of tubular, intermediate and punctate mitochondrial morphology in palmitate treated HCC1954 Pa (n = 5) and Lat (n = 6) cells. j. Western blot images showing expression of DRP1 along with p-DRP1S616and p-DRP1S637 in SKBR3 Pa and Lat cells respectively. In b and f-i, ‘n’ represents biologically independent samples and data presented as mean + /-SEM. P value in b, f, and h were calculated by two-tailed Unpaired t-test. The experiments shown in c, d, e, and j were repeated independently at least two or three times with similar results.

Source data

Extended Data Fig. 3 Latent cells oxidize internalized FAs and maintain redox homeostasis.

a. Mass spectrometry data showing DRP1 post-translational modifications including Serine phosphorylation (S616) and Lysine K10, K92, K160, K238 and K597 acetylation peaks in HCC1954 Lat cells. b. Western blots showing expression of carnitine palmitoyl-transferase 1 A (CPT1A) in HCC1954 (upper panel) and SKBR3 (lower panel) Pa and Lat cells. c and d. 13C16-Palmitic acid tracing showing enrichment of carnitine-conjugated fatty acids in HCC1954 and SKBR3 Pa and Lat cells. e-g. 13C16-Palmitic acid tracing showing labeled isotopologues of citrate, glutamate, and malate in SKBR3 Pa and Lat cells. In c-g, ‘n’ represents biologically independent samples and data presented as mean + /-SEM. P values in c-g were calculated by two-tailed Unpaired t-test. The experiments shown in b was repeated independently three times with similar results.

Source data

Extended Data Fig. 4 DRP1 promotes mitochondrial fragmentation in Latent cells.

a and b. 13C16-Palmitic acid tracing showing labeled isotopologues of GSSG and GSH in SKBR3 Pa and Lat cells. c. Measurement of steady-state GSH/GSSG ratio between Pa and Lat cells (HCC1954). a-c. n = 4, each group. d. Trypan blue exclusion assay showing viability of HCC1954 Pa and Lat cells when grown in palmitate (100 μM) for 48 hours. n = 5, each group. e. Immunofluorescence images showing localization of DRP1 to mitochondria in HCC1954 Pa and Lat cells. To visualize mitochondria Mitotracker Deep Red FM staining (500 nM, 30 min) was performed. f. Western blot images showing DRP1 and p-DRP1S616 in DRP1-depleted HCC1954 and SKBR3 Lat cells. g. Mitochondrial length quantification in Ctrl and DRP1-depleted HCC1954 Lat cells. n = 3, each group. h. Oil red O staining showing differential accumulation of LDs in Ctrl and DRP1-depleted HCC1954 Lat cells. i. Quantification of LDs in Ctrl vs DRP1 knockdown HCC1954 Lat cells upon palmitate treatment. n = 6, each group. j. Heatmap showing total fatty acid profiles of neutral lipid content generated by GC-MS in Ctrl and DRP1-KD Lat cells. Analysis was performed using 2.5×105 cells in each sample and data were normalized to internal fatty acid standards. n = 3, each group (Fig. 3g and Extended Data Fig. 4j were performed in a single experiment, thus controls are same). In a-d, g, i and j, ‘n’ represents biologically independent samples, data presented as mean + /-SEM and P values were calculated by two-tailed Unpaired t-test. The experiments shown in e, f and h were repeated independently at least two or three times with similar results.

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Extended Data Fig. 5 DRP1-driven mitochondrial dynamics enable FAO and redox homeostasis.

a. 13C16-Palmitic acid tracing showing enrichment of carnitine-conjugated fatty acids in DRP1-depleted HCC1954 Lat cells. b-g. LC-MS data showing enrichment of citrate, glutamate and malate isotopologues from 13C16-Palmitic acid in HCC1954 and SKBR3 Ctrl and DRP1-KD Lat cells respectively. h-k. Labeling of GSH and GSSG from 13C16-palmitate in HCC1954 and SKBR3 Lat cells (Ctrl and DRP1-KD). l. Measurement of relative steady-state GSH/GSSG ratio showing differences between Ctrl and DRP1-KD HCC1954 Lat cells (HCC1954). a-l. n = 4, each group. m. MTT assay showing cell viability of Ctrl (n = 8) and DRP1-KD (n = 8) HCC1954 Lat cells treated with palmitate(100 μM) for 48 hours (Fig. 3l and Extended Data Fig. 5m was performed in a single experiment with same control). n. Trypan blue exclusion assay showing viability of HCC1954 Pa and Lat cells grown in R3F + palmitic acid (100 μM) for 48 hours. n = 5, each group. o. Showing caspase-3 activity (Optical density at 405) in Ctrl and DRP1-depleted HCC1954 Lat cells. n = 4, each group. p and q. Oncosphere formation in Ctrl and DRP1-KD HCC1954 and SKBR3 Lat cells respectively. n = 8, each group. r. Showing the effect of treatment of N-acetyl-l-cysteine (NAC; 1 mM) on oncosphere forming ability of Ctrl and DRP1-depleted Lat cells. n = 8, each group. In a-r, ‘n’ represents biologically independent samples and data presented as mean + /-SEM. P values in a-q were calculated by two-tailed Unpaired t-test and r, was calculated by Ordinary one-way ANOVA.

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Extended Data Fig. 6 CPT1A aids FAO and altered mitochondrial dynamics in Latent cells.

a. Western blots showing validation of CPT1A knockdown in HCC1954 and SKBR3 Lat cells. b. Oil red O staining images showing differential accumulation of LDs in Ctrl and CPT1A-depleted Lat cells. c. Quantification of LDs in Ctrl and CPT1A knockdown Lat cells treated with sodium palmitate (100 µM) for 24 hours, n = 6, each group. d. Western blot images showing CPT1A, DRP1 and p-DRP1S616 in CPT1A and DRP1-depleted HCC1954 Lat cells. e. 13C16-Palmitic acid tracing showing enrichment of carnitine-conjugated fatty acids in Ctrl and CPT1A-depleted (Sh1, Sh2) HCC1954 Lat cells. f-k. LC-MS data showing labeling of citrate, glutamate, and malate isotopologues from 13C16-Palmitic acid in Ctrl and CPT1A-depleted HCC1954 and SKBR3 Lat cells. (Palmitate tracing in DRP1 and CTP1A depleted cells were performed using same controls). l-o. Showing fractions of GSH and GSSG isotopologues labeled from 13C16-palmitate in Ctrl and CPT1A-depleted HCC1954 and SKBR3 Lat cells. e-o. n = 4, each group. p and q. Showing the ability of oncosphere formation in Ctrl and CPT1A-depleted HCC1954 and SKBR3 Lat cells (n = 8) respectively. r. Orthotopic tumor generated from Ctrl (n = 10) and DRP1-depleted (n = 10) Lat cells. Tumors were collected 4 weeks post injection; tumor volume and weight were measured. s. Quantification of GFP + brain metastatic lesions in SKBR3 Lat cells (Ctrl and CPT1A-depleted knockdown; n = 4, each group). t. Western blot data showing rescue of DRP1 with doxycycline inducible overexpression of HA-tagged full-length DRP1 in DRP1-depleted cells Lat cells. In c, and e-q, ‘n’ represents biologically independent samples. r and s ‘n’ represent number of tumor and number of mice respectively. c, and e-s, data presented as mean + /-SEM. P values in c, e-q and s were calculated by Ordinary one-way ANOVA and r, two-tailed Unpaired t-test was used. The experiments shown in a, b, d, and t were repeated independently at least two or three times with similar results.

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Extended Data Fig. 7 DRP1 is essential for metachronous brain metastases.

a. Representative Kaplan-Meier plotter showing distant metastasis free survival for breast cancer patients with high or low expression of DNM1L (DRP1) gene. b. Western blot images showing expression of DRP1 and p-DRP1S616 in HCC1954 Pa, Lat and M-BM cells. c. Anti-TOMM20 antibody immunofluorescence images showing mitochondrial morphology of HCC1954 Pa, Lat, and M-BM cells. d. Classification of cells based on percentage distribution of tubular, intermediate and punctate mitochondrial morphology in HCC1954 Pa, Lat, and M-BM cells (n = 4, each group). e. Oncosphere formation in Ctrl and DRP1-KD HCC1954 M-BM cells. n = 8, each group. f. Showing the effect of treatment of N-acetyl-l-cysteine (NAC; 1 mM) on oncosphere forming ability of DRP1-depleted HCC1954 M-BM cells (n = 8, each group). g. Mice image with whole body photon flux showing metastatic burden in mice bearing HCC1954 Ctrl (n = 9) and DRP1(n = 9) depleted M-BM cells. h. Immunohistochemical (IHC) staining for cleaved caspase-3 (1:150, DAB, 10X) in Ctrl and DRP1-depleted M-BM cells injected mice brain section. i. Graph showing caspase-3 activity in Ctrl and DRP1-depleted HCC1954 M-BM cells. n = 4, each group. j. Orthotopic tumor generated from Ctrl and DRP1-depleted M-BM cells (n = 10, each group), post 4 weeks of cell injection. In d-f, and i, ‘n’ represents biologically independent samples, however g, and j ‘n’ represents number of mice and number of tumors respectively. In d-g, I, and j data presented as mean + /-SEM. P values in e, and j were calculated by two-tailed Unpaired t-test, f and g were calculated using Ordinary one-way ANOVA and two-tailed Mann–Whitney test respectively. The experiments shown in b, c, and h were repeated independently at least two to three times with similar results.

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Extended Data Fig. 8 Ectopic expression of DRP1 rescues DRP1-depleted phenotype.

a. Showing oncosphere image and quantification in Ctrl, DRP1 knockdown and DRP1-rescued HCC1954 Lat cells (n = 6, each group). Data presented as mean + /-SEM. P values were calculated using Ordinary one-way ANOVA. b. Whole body image and photon flux showing metastatic burden in mice bearing Ctrl (n = 10), DRP1-depleted (n = 9) and DRP1-rescued (n = 9) M-BM cells. Data presented as mean + /-SEM. P value was calculated by Kruskal–Wallis test. In a ‘n’ represents biologically independent samples and b ‘n’ represents number of mice.

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Extended Data Fig. 9 DRP1 inhibitors increases LDs and decreases survival of Latent and M-BM cells.

a. IF images of HCC1954 Pa, Lat, and M-BM cells with or without Mdivi-1 treatment (12.5 µM, 48 hours). b. Quantification of number of LDs between Ctrl and Mdivi-1 treated HCC1954 Pa, Lat, and M-BM (n = 5, each group) cells. c. Showing viability of HCC1954 Pa, Lat, and M-BM (n = 8, each group) cells upon Mdivi-1 treatment (0, 3.125, 6.25,12.5 and 25 µM) for 48 hours. d. IF images of SKBR3 Pa, Lat, and M-BM cells with or without Mdivi-1 treatment (12.5 µM, 48 hours). e. Quantification of number of LDs in SKBR3 Pa, Lat, and M-BM (n = 5, each group) cells after Mdivi-1 treatment. f. Showing viability of HCC1954 Pa, Lat, and M-BM (n = 8, each group) cells upon Mdivi-1 treatment (0, 3.125, 6.25,12.5 and 25 µM) for 48 hours. g. IF images of Ctrl and Dynasore treated HCC1954 Pa, Lat, and M-BM cells. h. Quantification of number of LDs in Ctrl and Dynasore treated (25 µM) HCC1954 Pa, Lat, and M-BM (n = 3, each group) cells. i. Showing viability of HCC1954 Pa, Lat, and M-BM (n = 8, each group) cells upon treatment of Dynasore (0, 5, 10,25 and 50 µM) for 48 hours. In b, c, e, f, h and i, ‘n’ represents biologically independent samples and data presented as mean + /-SEM and P values were calculated by Ordinary one-way ANOVA. The experiments shown in a, d, and g were repeated independently two times with similar results.

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Extended Data Fig. 10 Pharmacologic inhibition of DRP1 attenuates brain metastasis.

a. Mice body weight comparison after treatment with vehicle (10% DMSO in corn oil, n = 7) and Mdivi-1(40 mg/kg, n = 7) for 4 weeks. Data presented as mean + /-SEM, Unpaired t-test. b and c. Mice image with whole body, spine and brain-only photon flux showing metastatic burden in mice bearing HCC1954 M-BM cells treated with vehicle (10% DMSO in corn oil, n = 8) and Mdivi-1(40 mg/kg, n = 7). Data presented as mean + /-SEM. P value was calculated by Mann–Whitney test. d and e. Bar graph showing ability of HCC1954 and SKBR3 Pa, Lat, and M-BM to form oncospheres in the presence of HER2 TKIs (lapatinib (2 μM) and tucatinib (3 μM)) and DRP1 inhibitor Mdivi-1 (5 μM) alone or in combination. ‘n’ of individual group has been provided in source data file. ‘n’ in a and c represents number of mice however in d and e it represents biologically independent samples. Dotted line indicates control. Data presented as mean + /-SEM. P values were calculated by One-way ANOVA by comparing all groups to control.

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Supplementary information

Supplementary Information

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Reporting Summary

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Supplementary Video 1

Confocal microscope time-lapse imaging showing lipid transfer from astrocytes to HCC1954 Pa cells in co-culture setting (bright field, astrocytes; green, Pa cells; red, BODIPY-C12).

Supplementary Video 2

Confocal microscope time-lapse imaging showing lipid transfer from astrocytes to HCC1954 Lat cells in co-culture setting (bright field, astrocytes; green, Lat cells; red, BODIPY-C12).

Supplementary Video 3

IncuCyte3 time-lapse images showing lipid uptake and cell death in HCC1954 Pa cells cultured with 2 μM BODIPY-558/568-C12 (bright field, Pa cells; red, BODIPY-C12).

Supplementary Video 4

IncuCyte3 time-lapse images showing lipid uptake and cell death in HCC1954 Lat cells cultured with 2 μM BODIPY-558/568-C12 (bright field, Lat cells; red, BODIPY-C12).

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Parida, P.K., Marquez-Palencia, M., Ghosh, S. et al. Limiting mitochondrial plasticity by targeting DRP1 induces metabolic reprogramming and reduces breast cancer brain metastases. Nat Cancer 4, 893–907 (2023). https://doi.org/10.1038/s43018-023-00563-6

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