Breast Cancer Research and Treatment

, Volume 178, Issue 1, pp 75–86 | Cite as

PK-M2-mediated metabolic changes in breast cancer cells induced by ionizing radiation

  • Le Zhang
  • Justine Bailleul
  • Taha Yazal
  • Kevin Dong
  • David Sung
  • Amy Dao
  • Laura Gosa
  • David Nathanson
  • Kruttika Bhat
  • Sara Duhachek-Muggy
  • Claudia Alli
  • Milana Bochkur Dratver
  • Frank Pajonk
  • Erina VlashiEmail author
Preclinical study



Radiotherapy (RT) constitutes an important part of breast cancer treatment. However, triple negative breast cancers (TNBC) exhibit remarkable resistance to most therapies, including RT. Developing new ways to radiosensitize TNBC cells could result in improved patient outcomes. The M2 isoform of pyruvate kinase (PK-M2) is believed to be responsible for the re-wiring of cancer cell metabolism after oxidative stress. The aim of the study was to determine the effect of ionizing radiation (IR) on PK-M2-mediated metabolic changes in TNBC cells, and their survival. In addition, we determine the effect of PK-M2 activators on breast cancer stem cells, a radioresistant subpopulation of breast cancer stem cells.


Glucose uptake, lactate production, and glutamine consumption were assessed. The cellular localization of PK-M2 was evaluated by western blot and confocal microscopy. The small molecule activator of PK-M2, TEPP46, was used to promote its pyruvate kinase function. Finally, effects on cancer stem cell were evaluated via sphere forming capacity.


Exposure of TNBC cells to IR increased their glucose uptake and lactate production. As expected, PK-M2 expression levels also increased, especially in the nucleus, although overall pyruvate kinase activity was decreased. PK-M2 nuclear localization was shown to be associated with breast cancer stem cells, and activation of PK-M2 by TEPP46 depleted this population.


Radiotherapy can induce metabolic changes in TNBC cells, and these changes seem to be mediated, at least in part by PK-M2. Importantly, our results show that activators of PK-M2 can deplete breast cancer stem cells in vitro. This study supports the idea of combining PK-M2 activators with radiation to enhance the effect of radiotherapy in resistant cancers, such as TNBC.


Pyruvate kinase Radiation therapy Breast cancer Metabolism 







Breast cancer


Fibroblast growth factor 2


Cancer stem cell


Epidermal growth factor


Glyceraldehyde 3-phosphate dehydrogenase




2′,7′-dichlorodihydrofluorescein diacetate


Hypoxia inducible factor


Ionizing radiation


Nicotinamide adenine dinucleotide phosphate


M2 isoform of pyruvate kinase


Pentose phosphate pathway


Reactive oxygen species


Radiation therapy


Triple negative breast cancer



We would like to acknowledge Dr. William McBride for his careful editing of the manuscript and thoughtful feedback and comments.

Author contributions

LZ and JB performed most of the experiments and data analysis and assisted with editing of the manuscript. TY, KD, DS, AD, LG, DN, KB, CA, and MBD assisted with experiments and data analysis. FP assisted with experimental design and editing of the manuscript. EV designed all experiments, oversaw data analysis, and wrote the manuscript.


EV was supported by a Junior Faculty Award (JFA) from the American Society for Radiation Oncology (ASTRO) and the UCLA SPORE in Brain Cancer (P50 CA211015). FP was supported by grants from the National Cancer Institute (CA137110, CA161294).

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflicts of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10549_2019_5376_MOESM1_ESM.docx (4.6 mb)
Supplementary material 1 (DOCX 4759 kb)


  1. 1.
    Sjostrom M, Lundstedt D, Hartman L, Holmberg E, Killander F, Kovacs A, Malmstrom P, Nimeus E, Werner Ronnerman E, Ferno M et al (2017) Response to radiotherapy after breast-conserving surgery in different breast cancer subtypes in the swedish breast cancer group 91 radiotherapy randomized clinical trial. J Clin Oncol 35:3222–3229PubMedGoogle Scholar
  2. 2.
    Liedtke C, Mazouni C, Hess KR, Andre F, Tordai A, Mejia JA, Symmans WF, Gonzalez-Angulo AM, Hennessy B, Green M et al (2008) Response to neoadjuvant therapy and long-term survival in patients with triple-negative breast cancer. J Clin Oncol 26(8):1275–1281Google Scholar
  3. 3.
    Vlashi E, Pajonk F (2014) Cancer stem cells, cancer cell plasticity and radiation therapy. Semin Cancer Biol 31:28–35PubMedGoogle Scholar
  4. 4.
    Pajonk F, Vlashi E, McBride WH (2010) Radiation resistance of cancer stem cells: the 4 R’s of radiobiology revisited. Stem cells (Dayton, Ohio) 28(4):639–648Google Scholar
  5. 5.
    Phillips TM, McBride WH, Pajonk F (2006) The response of CD24(-/low)/CD44 + breast cancer-initiating cells to radiation. J Natl Cancer Inst 98(24):1777–1785PubMedGoogle Scholar
  6. 6.
    Lagadec C, Vlashi E, Della Donna L, Dekmezian C, Pajonk F (2012) Radiation-induced reprogramming of breast cancer cells. Stem Cells (Dayton, Ohio) 30(5):833–844Google Scholar
  7. 7.
    Lagadec C, Vlashi E, Della Donna L, Meng Y, Dekmezian C, Kim K, Pajonk F (2010) Survival and self-renewing capacity of breast cancer initiating cells during fractionated radiation treatment. Breast Cancer Res 12(1):R13PubMedPubMedCentralGoogle Scholar
  8. 8.
    Woodward WA, Chen MS, Behbod F, Alfaro MP, Buchholz TA, Rosen JM (2007) WNT/beta-catenin mediates radiation resistance of mouse mammary progenitor cells. Proc Natl Acad Sci USA 104(2):618–623PubMedGoogle Scholar
  9. 9.
    O’Neill P, Wardman P (2009) Radiation chemistry comes before radiation biology. Int J Radiat Biol 85(1):9–25PubMedGoogle Scholar
  10. 10.
    Liou GY, Storz P (2010) Reactive oxygen species in cancer. Free Radical Res 44(5):479–496Google Scholar
  11. 11.
    Colussi C, Albertini MC, Coppola S, Rovidati S, Galli F, Ghibelli L (2000) H2O2-induced block of glycolysis as an active ADP-ribosylation reaction protecting cells from apoptosis. FASEB J 14(14):2266–2276PubMedGoogle Scholar
  12. 12.
    Ralser M, Wamelink MM, Kowald A, Gerisch B, Heeren G, Struys EA, Klipp E, Jakobs C, Breitenbach M, Lehrach H et al (2007) Dynamic rerouting of the carbohydrate flux is key to counteracting oxidative stress. J Biol 6(4):10PubMedPubMedCentralGoogle Scholar
  13. 13.
    Ralser M, Wamelink MM, Latkolik S, Jansen EE, Lehrach H, Jakobs C (2009) Metabolic reconfiguration precedes transcriptional regulation in the antioxidant response. Nat Biotechnol 27(7):604–605PubMedGoogle Scholar
  14. 14.
    Anastasiou D, Poulogiannis G, Asara JM, Boxer MB, Jiang JK, Shen M, Bellinger G, Sasaki AT, Locasale JW, Auld DS et al (2011) Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. Science (New York, NY) 334(6060):1278–1283Google Scholar
  15. 15.
    Eigenbrodt E, Reinacher M, Scheefers-Borchel U, Scheefers H, Friis R (1992) Double role for pyruvate kinase type M2 in the expansion of phosphometabolite pools found in tumor cells. Crit Rev Oncog 3(1–2):91–115PubMedGoogle Scholar
  16. 16.
    Chaneton B, Gottlieb E (2012) Rocking cell metabolism: revised functions of the key glycolytic regulator PKM2 in cancer. Trends Biochem Sci 37(8):309–316PubMedGoogle Scholar
  17. 17.
    Dayton TL, Jacks T, Vander Heiden MG (2016) PKM2, cancer metabolism, and the road ahead. EMBO Rep 17(12):1721–1730PubMedPubMedCentralGoogle Scholar
  18. 18.
    Zhao Z, Song Z, Liao Z, Liu Z, Sun H, Lei B, Chen W, Dang C (2016) PKM2 promotes stemness of breast cancer cell by through Wnt/beta-catenin pathway. Tumour Biol 37(3):4223–4234PubMedGoogle Scholar
  19. 19.
    Warburg O (1924) On the metabolism of carcinoma cells. Biochem Z 152(309–344):309Google Scholar
  20. 20.
    Vander Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science (New York, NY) 324(5930):1029–1033Google Scholar
  21. 21.
    Christofk HR, Vander Heiden MG, Wu N, Asara JM, Cantley LC (2008) Pyruvate kinase M2 is a phosphotyrosine-binding protein. Nature 452(7184):181–186Google Scholar
  22. 22.
    Christofk HR, Vander Heiden MG, Harris MH, Ramanathan A, Gerszten RE, Wei R, Fleming MD, Schreiber SL, Cantley LC (2008) The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 452(7184):230–233PubMedPubMedCentralGoogle Scholar
  23. 23.
    Gao X, Wang H, Yang JJ, Liu X, Liu ZR (2012) Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase. Mol Cell 45(5):598–609PubMedPubMedCentralGoogle Scholar
  24. 24.
    Lee J, Kim HK, Han YM, Kim J (2008) Pyruvate kinase isozyme type M2 (PKM2) interacts and cooperates with Oct-4 in regulating transcription. Int J Biochem Cell Biol 40(5):1043–1054PubMedGoogle Scholar
  25. 25.
    Yang W, Lu Z (2013) Nuclear PKM2 regulates the Warburg effect. Cell Cycle (Georgetown, Tex) 12(19):3154–3158Google Scholar
  26. 26.
    Yang W, Xia Y, Ji H, Zheng Y, Liang J, Huang W, Gao X, Aldape K, Lu Z (2011) Nuclear PKM2 regulates beta-catenin transactivation upon EGFR activation. Nature 480(7375):118–122PubMedPubMedCentralGoogle Scholar
  27. 27.
    Anastasiou D, Yu Y, Israelsen WJ, Jiang JK, Boxer MB, Hong BS, Tempel W, Dimov S, Shen M, Jha A et al (2012) Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis. Nat Chem Biol 8(10):839–847PubMedPubMedCentralGoogle Scholar
  28. 28.
    Boxer MB, Jiang JK, Vander Heiden MG, Shen M, Skoumbourdis AP, Southall N, Veith H, Leister W, Austin CP, Park HW et al (2010) Evaluation of substituted N, N’-diarylsulfonamides as activators of the tumor cell specific M2 isoform of pyruvate kinase. J Med Chem 53(3):1048–1055PubMedPubMedCentralGoogle Scholar
  29. 29.
    Kuehne A, Emmert H, Soehle J, Winnefeld M, Fischer F, Wenck H, Gallinat S, Terstegen L, Lucius R, Hildebrand J et al (2015) Acute activation of oxidative pentose phosphate pathway as first-line response to oxidative stress in human skin cells. Mol Cell 59(3):359–371PubMedGoogle Scholar
  30. 30.
    Le Goffe C, Vallette G, Charrier L, Candelon T, Bou-Hanna C, Bouhours JF, Laboisse CL (2002) Metabolic control of resistance of human epithelial cells to H2O2 and NO stresses. Biochem J 364(Pt 2):349–359PubMedPubMedCentralGoogle Scholar
  31. 31.
    Vlashi E, Lagadec C, Vergnes L, Reue K, Frohnen P, Chan M, Alhiyari Y, Dratver MB, Pajonk F (2014) Metabolic differences in breast cancer stem cells and differentiated progeny. Breast Cancer Res Treat 146(3):525–534PubMedPubMedCentralGoogle Scholar
  32. 32.
    Vlashi E, Lagadec C, Vergnes L, Matsutani T, Masui K, Poulou M, Popescu R, Della Donna L, Evers P, Dekmezian C et al (2011) Metabolic state of glioma stem cells and nontumorigenic cells. Proc Natl Acad Sci USA 108(38):16062–16067PubMedGoogle Scholar
  33. 33.
    Vlashi E, Kim K, Lagadec C, Donna LD, McDonald JT, Eghbali M, Sayre JW, Stefani E, McBride W, Pajonk F (2009) In vivo imaging, tracking, and targeting of cancer stem cells. J Natl Cancer Inst 101(5):350–359PubMedPubMedCentralGoogle Scholar
  34. 34.
    Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS (2003) In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17(10):1253–1270PubMedPubMedCentralGoogle Scholar
  35. 35.
    Jin L, Alesi GN, Kang S (2016) Glutaminolysis as a target for cancer therapy. Oncogene 35(28):3619–3625PubMedGoogle Scholar
  36. 36.
    Warburg O, Wind F, Negelein E (1927) The metabolism of tumors in the body. J Gen Physiol 8(6):519–530PubMedPubMedCentralGoogle Scholar
  37. 37.
    Walsh MJ, Brimacombe KR, Anastasiou D, Yu Y, Israelsen WJ, Hong BS, Tempel W, Dimov S, Veith H, Yang H et al (2010) ML265: a potent PKM2 activator induces tetramerization and reduces tumor formation and size in a mouse xenograft model. In: Probe Reports from the NIH Molecular Libraries Program. BethesdaGoogle Scholar
  38. 38.
    Lv L, Xu YP, Zhao D, Li FL, Wang W, Sasaki N, Jiang Y, Zhou X, Li TT, Guan KL et al (2013) Mitogenic and oncogenic stimulation of K433 acetylation promotes PKM2 protein kinase activity and nuclear localization. Mol Cell 52(3):340–352PubMedPubMedCentralGoogle Scholar
  39. 39.
    Hu Y, Smyth GK (2009) ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods 347(1–2):70–78PubMedGoogle Scholar
  40. 40.
  41. 41.
    Adikrisna R, Tanaka S, Muramatsu S, Aihara A, Ban D, Ochiai T, Irie T, Kudo A, Nakamura N, Yamaoka S et al (2012) Identification of pancreatic cancer stem cells and selective toxicity of chemotherapeutic agents. Gastroenterology 143(1):234–245PubMedGoogle Scholar
  42. 42.
    Della Donna L, Lagadec C, Pajonk F (2012) Radioresistance of prostate cancer cells with low proteasome activity. Prostate 72(8):868–874PubMedGoogle Scholar
  43. 43.
    Hayashi K, Tamari K, Ishii H, Konno M, Nishida N, Kawamoto K, Koseki J, Fukusumi T, Kano Y, Nishikawa S et al (2014) Visualization and characterization of cancer stem-like cells in cervical cancer. Int J Oncol 45:2468–2474PubMedGoogle Scholar
  44. 44.
    Lagadec C, Vlashi E, Bhuta S, Lai C, Mischel P, Werner M, Henke M, Pajonk F (2014) Tumor cells with low proteasome subunit expression predict overall survival in head and neck cancer patients. BMC Cancer 14:152PubMedPubMedCentralGoogle Scholar
  45. 45.
    Munakata K, Uemura M, Tanaka S, Kawai K, Kitahara T, Miyo M, Kano Y, Nishikawa S, Fukusumi T, Takahashi Y et al (2016) Cancer stem-like properties in colorectal cancer cells with low proteasome activity. Clinical Cancer Res 22(21):5277–5286Google Scholar
  46. 46.
    Muramatsu S, Tanaka S, Mogushi K, Adikrisna R, Aihara A, Ban D, Ochiai T, Irie T, Kudo A, Nakamura N et al (2013) Visualization of stem cell features in human hepatocellular carcinoma reveals in vivo significance of tumor-host interaction and clinical course. Hepatology 58(1):218–228PubMedGoogle Scholar
  47. 47.
    Pan J, Zhang Q, Wang Y, You M (2010) 26S proteasome activity is down-regulated in lung cancer stem-like cells propagated in vitro. PLoS ONE 5(10):e13298PubMedPubMedCentralGoogle Scholar
  48. 48.
    Stacer AC, Wang H, Fenner J, Dosch JS, Salomonnson A, Luker KE, Luker GD, Rehemtulla A, Ross BD (2015) Imaging reporters for proteasome activity identify tumor- and metastasis-initiating cells. Mol Imaging 14:414–428PubMedPubMedCentralGoogle Scholar
  49. 49.
    Tamari K, Hayashi K, Ishii H, Kano Y, Konno M, Kawamoto K, Nishida N, Koseki J, Fukusumi T, Hasegawa S et al (2014) Identification of chemoradiation-resistant osteosarcoma stem cells using an imaging system for proteasome activity. Int J Oncol 45:2349–2354PubMedGoogle Scholar
  50. 50.
    Tang B, Raviv A, Esposito D, Flanders KC, Daniel C, Nghiem BT, Garfield S, Lim L, Mannan P, Robles AI et al (2015) A flexible reporter system for direct observation and isolation of cancer stem cells. Stem cell Rep 4(1):155–169Google Scholar
  51. 51.
    Vlashi E, Lagadec C, Chan M, Frohnen P, McDonald AJ, Pajonk F (2013) Targeted elimination of breast cancer cells with low proteasome activity is sufficient for tumor regression. Breast Cancer Res Treat 141(2):197–203PubMedGoogle Scholar
  52. 52.
    Grant CM (2008) Metabolic reconfiguration is a regulated response to oxidative stress. J Biol 7(1):1PubMedPubMedCentralGoogle Scholar
  53. 53.
    Warburg O (1925) On the formation of lactic acid with growth. Biochem Z 160:307–311Google Scholar
  54. 54.
    Tamada M, Suematsu M, Saya H (2012) Pyruvate kinase M2: multiple faces for conferring benefits on cancer cells. Clin Cancer Res 18(20):5554–5561PubMedGoogle Scholar
  55. 55.
    Jurica MS, Mesecar A, Heath PJ, Shi W, Nowak T, Stoddard BL (1998) The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate. Structure 6(2):195–210PubMedGoogle Scholar
  56. 56.
    Chaneton B, Hillmann P, Zheng L, Martin ACL, Maddocks ODK, Chokkathukalam A, Coyle JE, Jankevics A, Holding FP, Vousden KH et al (2012) Serine is a natural ligand and allosteric activator of pyruvate kinase M2. Nature 491(7424):458–462PubMedPubMedCentralGoogle Scholar
  57. 57.
    Luo W, Hu H, Chang R, Zhong J, Knabel M, O’Meally R, Cole RN, Pandey A, Semenza GL (2011) Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1. Cell 145(5):732–744PubMedPubMedCentralGoogle Scholar
  58. 58.
    Wang HJ, Hsieh YJ, Cheng WC, Lin CP, Lin YS, Yang SF, Chen CC, Izumiya Y, Yu JS, Kung HJ et al (2014) JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1alpha-mediated glucose metabolism. Proc Natl Acad Sci USA 111(1):279–284PubMedGoogle Scholar
  59. 59.
    Yu X, Li S (2017) Non-metabolic functions of glycolytic enzymes in tumorigenesis. Oncogene 36(19):2629–2636PubMedGoogle Scholar
  60. 60.
    Cheng TY, Yang YC, Wang HP, Tien YW, Shun CT, Huang HY, Hsiao M, Hua KT (2018) Pyruvate kinase M2 promotes pancreatic ductal adenocarcinoma invasion and metastasis through phosphorylation and stabilization of PAK2 protein. OncogeneGoogle Scholar
  61. 61.
    Hosios AM, Fiske BP, Gui DY, Vander Heiden MG (2015) Lack of evidence for PKM2 protein kinase activity. Mol Cell 59(5):850–857PubMedPubMedCentralGoogle Scholar
  62. 62.
    Sizemore ST, Zhang M, Cho JH, Sizemore GM, Hurwitz B, Kaur B, Lehman NL, Ostrowski MC, Robe PA, Miao W et al (2018) Pyruvate kinase M2 regulates homologous recombination-mediated DNA double-strand break repair. Cell Res 28:1090PubMedPubMedCentralGoogle Scholar
  63. 63.
    Palsson-McDermott EM, Curtis AM, Goel G, Lauterbach MAR, Sheedy FJ, Gleeson LE, van den Bosch MWM, Quinn SR, Domingo-Fernandez R, Johnston DGW et al (2015) Pyruvate kinase M2 regulates Hif-1alpha activity and IL-1beta induction and is a critical determinant of the warburg effect in LPS-activated macrophages. Cell Metab 21(2):347PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Le Zhang
    • 1
  • Justine Bailleul
    • 1
  • Taha Yazal
    • 1
  • Kevin Dong
    • 1
  • David Sung
    • 1
  • Amy Dao
    • 1
  • Laura Gosa
    • 2
  • David Nathanson
    • 2
    • 3
  • Kruttika Bhat
    • 1
  • Sara Duhachek-Muggy
    • 1
  • Claudia Alli
    • 1
  • Milana Bochkur Dratver
    • 1
  • Frank Pajonk
    • 1
    • 3
  • Erina Vlashi
    • 1
    • 3
    Email author
  1. 1.Department of Radiation OncologyDavid Geffen School of Medicine at UCLALos AngelesUSA
  2. 2.Department of Molecular and Medical Pharmacology, David Geffen School of MedicineUniversity of California, Los AngelesLos AngelesUSA
  3. 3.Jonsson Comprehensive Cancer CenterUniversity of California, Los AngelesLos AngelesUSA

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