Skip to main content

Advertisement

SpringerLink
Log in

Circadian Clock Gene bmal1 Acts as a Tumor Suppressor Gene in a Mice Model of Human Glioblastoma

  • Published:
Molecular Neurobiology Aims and scope Submit manuscript

Abstract

Glioblastomas derived from malignant astrocytes are the most common primary tumors of the central nervous system in humans, exhibiting very bad prognosis. Treatment with surgery, radiotherapy, and chemotherapy (mainly using temozolomide), generates as much one-year survival. The circadian clock controls different aspects of tumor development, and its role in GBM is beginning to be explored. Here, the role of the canonic circadian clock gene bmal1 was studied in vivo in a nude mice model bearing human GBMs from LN229 cells xenografted orthotopically in the dorsal striatum. For that aim, a bmal1 knock-down was generated in LN229 cells by CRISPR/Cas9 gene editing tool, and tumor progression was followed in male mice by measuring survival, tumor growth, cell proliferation and prognosis with CD44 marker, as well as astrocyte activation in the tumor microenvironment with GFAP and nestin markers. Disruption of bmal1 in the tumor decreased survival, increased tumor growth and CD44 expression, worsened motor performance, as well as increased GFAP expression in astrocytes at tumor microenvironment. In addition, survival and tumor progression was not affected in mice bearing LN229 wild type GBM that underwent circadian disruption by constant light, as compared to mice synchronized to 12:12 light–dark cycles. These results consistently demonstrate in an in vivo orthotopic model of human GBM, that bmal1 has a key role as a tumor suppressor gene regulating GBM progression.

Graphical Abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

Data Availability

The datasets generated during and/or analyzed during the current study are not publicly available due to Laboratory Policies, but are available from the corresponding author on reasonable request.

References

  1. Omuro A, De Angelis LM (2013) Glioblastoma and other malignant gliomas: a clinical review. JAMA - J Am Med Assoc. https://doi.org/10.1001/jama.2013.280319

    Article  Google Scholar 

  2. Phillips HS et al (2006) Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. Cancer Cell. https://doi.org/10.1016/j.ccr.2006.02.019

    Article  PubMed  Google Scholar 

  3. Stupp R et al (2009) Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 10:459–466

    Article  PubMed  CAS  Google Scholar 

  4. Golombek DA, Rosenstein RE (2010) Physiology of Circadian Entrainment. Physiol Rev. https://doi.org/10.1152/physrev.00009.2009

    Article  PubMed  Google Scholar 

  5. Hastings MH, Maywood ES, Brancaccio M (2018) Generation of circadian rhythms in the suprachiasmatic nucleus. Nat Rev Neurosci 19(8):453–469. https://doi.org/10.1038/s41583-018-0026-z

    Article  PubMed  CAS  Google Scholar 

  6. Takahashi JS (2015) Molecular components of the circadian clock in mammals. Diabetes Obes Metab 17(Suppl 1):6–11. https://doi.org/10.1111/dom.12514

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Bunger MK et al (2000) Mop3 is an essential component of the master circadian pacemaker in mammals. Cell 103(7):1009–1017. https://doi.org/10.1016/s0092-8674(00)00205-1

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Zhao C et al (2022) Circadian clock gene BMAL1 inhibits the proliferation and tumor-formation ability of nasopharyngeal carcinoma cells and increases the sensitivity of radiotherapy. Chronobiol Int 39(10):1340–1351. https://doi.org/10.1080/07420528.2022.2105708

    Article  PubMed  CAS  Google Scholar 

  9. Jiang W et al (2016) The circadian clock gene Bmal1 acts as a potential anti-oncogene in pancreatic cancer by activating the p53 tumor suppressor pathway. Cancer Lett 371(2):314–325. https://doi.org/10.1016/j.canlet.2015.12.002

    Article  PubMed  CAS  Google Scholar 

  10. Qu M et al (2023) Circadian regulator BMAL1: CLOCK promotes cell proliferation in hepatocellular carcinoma by controlling apoptosis and cell cycle. Proc Natl Acad Sci U S A 120(2):e2214829120. https://doi.org/10.1073/pnas.2214829120

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Wang J et al (2019) Circadian protein BMAL1 promotes breast cancer cell invasion and metastasis by up-regulating matrix metalloproteinase9 expression. Cancer Cell Int 19:182. https://doi.org/10.1186/s12935-019-0902-2

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Dong P et al (2022) BMAL1 induces colorectal cancer metastasis by stimulating exosome secretion. Mol Biol Rep 49(1):373–384. https://doi.org/10.1007/s11033-021-06883-z

    Article  PubMed  CAS  Google Scholar 

  13. Wang D et al (2023) Identification and characterization of the CDK1-BMAL1-UHRF1 pathway driving tumor progression. iScience 26(4):106544. https://doi.org/10.1016/j.isci.2023.106544

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Masri S, Cervantes M, Sassone-Corsi P (2013) The circadian clock and cell cycle: interconnected biological circuits. Curr Opin Cell Biol 25(6):730–734. https://doi.org/10.1016/j.ceb.2013.07.013

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Fu XJ et al (2016) The important tumor suppressor role of PER1 in regulating the cyclin–CDK–CKI network in SCC15 human oral squamous cell carcinoma cells. Onco Targets Ther https://doi.org/10.2147/OTT.S100952

  16. Gery S et al (2006) The circadian gene per1 plays an important role in cell growth and DNA damage control in human cancer cells. Mol Cell 22(3):375–382

    Article  PubMed  CAS  Google Scholar 

  17. Chen ST et al (2005) Deregulated expression of the PER1, PER2 and PER3 genes in breast cancers. Carcinogenesis https://doi.org/10.1093/carcin/bgi075

  18. Zhao H et al (2014) Prognostic relevance of Period1 (Per1) and Period2 (Per2) expression in human gastric cancer. Int J Clin Exp Pathol 7(2):619–30

  19. Hsu CM et al (2012) Altered expression of circadian clock genes in head and neck squamous cell carcinoma. Tumor Biol 33(1):149–55. https://doi.org/10.1007/s13277-011-0258-2

  20. Farshadi E et al (2019) The positive circadian regulators CLOCK and BMAL1 control G2/M cell cycle transition through Cyclin B1. Cell Cycle 18(1):16–33. https://doi.org/10.1080/15384101.2018.1558638

  21. Matsuo T et al (2003) Control mechanism of the circadian clock for timing of cell division in vivo. Science 302(5643):255–9. https://doi.org/10.1126/science.1086271

  22. Gwon DH et al (2020) BMAL1 suppresses proliferation, migration, and invasion of U87MG cells by downregulating cyclin B1, Phospho-AKT, and Metalloproteinase-9. Int J Mol Sci 21(7):2352. https://doi.org/10.3390/ijms21072352

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Jung CH et al (2013) Bmal1 suppresses cancer cell invasion by blocking the phosphoinositide 3-kinase-Akt-MMP-2 signaling pathway. Oncol Rep 29(6):2109–2113. https://doi.org/10.3892/or.2013.2381

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Wagner PM et al (2021) Temporal regulation of tumor growth in nocturnal mammals: in vivo studies and chemotherapeutical potential. FASEB J 35(2):e21231. https://doi.org/10.1096/fj.202001753R

    Article  PubMed  CAS  Google Scholar 

  25. Dong Z et al (2019) Targeting glioblastoma stem cells through disruption of the circadian clock. Cancer Discov 9(11):1556–1573. https://doi.org/10.1158/2159-8290.CD-19-0215

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Pang L et al (2023) Circadian regulator CLOCK promotes tumor angiogenesis in glioblastoma. Cell Rep 42(2):112127. https://doi.org/10.1016/j.celrep.2023.112127

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Xuan W et al (2022) Circadian Regulator CLOCK Drives Immunosuppression in Glioblastoma. Cancer Immunol Res 10(6):770–784. https://doi.org/10.1158/2326-6066.CIR-21-0559

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Filipski E et al (2004) Effects of chronic jet lag on tumor progression in mice. Cancer Res 64(21):7879–7885

    Article  PubMed  CAS  Google Scholar 

  29. Aiello I et al (2020) Circadian disruption promotes tumor-immune microenvironment remodeling favoring tumor cell proliferation. Sci Adv. 6(42):eaaz4530. https://doi.org/10.1126/sciadv

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Hadadi E et al (2020) Chronic circadian disruption modulates breast cancer stemness and immune microenvironment to drive metastasis in mice. Nat Commun 11(1):3193. https://doi.org/10.1038/s41467-020-16890-6

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Wagner PM et al (2019) Proliferative glioblastoma cancer cells exhibit persisting temporal control of metabolism and display differential temporal drug susceptibility in chemotherapy. Mol Neurobiol 56(2):1276–1292. https://doi.org/10.1007/s12035-018-1152-3

    Article  PubMed  CAS  Google Scholar 

  32. Trebucq LL et al (2021) Timing of novel Drug 1A–116 to circadian rhythms improves therapeutic effects against glioblastoma. Pharmaceutics 13(7):1091. https://doi.org/10.3390/pharmaceutics13071091

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Montgomery CA Jr (1990) Oncological and toxicological research: Alleviation and control of pain and distress in laboratory animals. Cancer Bulletin 42:230–237

  34. Magno LAV, Collodetti M, Tenza-Ferrer H, Romano-Silva MA (2019) Cylinder test to assess sensory-motor function in a mouse model of Parkinson’s disease. Bio Protoc 9(16):e3337. https://doi.org/10.21769/BioProtoc.3337

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. King GD et al (2008) Flt3L in combination with HSV1-TK-mediated gene therapy reverses brain tumor-induced behavioral deficits. Mol Ther 16(4):682–690. https://doi.org/10.1038/mt.2008.18

    Article  PubMed  CAS  Google Scholar 

  36. Wu G et al (2016) MetaCycle: an integrated R package to evaluate periodicity in large scale data. Bioinformatics. https://doi.org/10.1093/bioinformatics/btw405

    Article  PubMed  PubMed Central  Google Scholar 

  37. El-Athman R, Fuhr L, Relógio A (2018) A systems-level analysis reveals circadian regulation of splicing in colorectal cancer. EBioMedicine 33:68–81. https://doi.org/10.1016/j.ebiom.2018.06.012

    Article  PubMed  PubMed Central  Google Scholar 

  38. Xiang R et al (2018) Circadian clock gene Per2 downregulation in non-small cell lung cancer is associated with tumor progression and metastasis. Oncol Rep 40(5):3040–3048. https://doi.org/10.3892/or.2018.6704

    Article  PubMed  CAS  Google Scholar 

  39. Mooney KL et al (2016) The role of CD44 in glioblastoma multiforme. J Clin Neurosci 34:1–5. https://doi.org/10.1016/j.jocn.2016.05.012

    Article  PubMed  CAS  Google Scholar 

  40. Yu M, Li W, Wang Q, Wang Y, Lu F (2018) Circadian regulator NR1D2 regulates glioblastoma cell proliferation and motility. Oncogene 37(35):4838–4853. https://doi.org/10.1038/s41388-018-0319-8

    Article  PubMed  CAS  Google Scholar 

  41. Dzobo K et al (2020) Advances in therapeutic targeting of cancer stem cells within the tumor microenvironment: an updated review. Cells 9(8):1896. https://doi.org/10.3390/cells9081896

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Hou C et al (2019) Overexpression of CD44 is associated with a poor prognosis in grade II/III gliomas. J Neurooncol 145(2):201–210. https://doi.org/10.1007/s11060-019-03288-8

    Article  PubMed  CAS  Google Scholar 

  43. Ameneiro C et al (2020) BMAL1 coordinates energy metabolism and differentiation of pluripotent stem cells. Life Sci Alliance 3(5):e201900534. https://doi.org/10.26508/lsa.201900534

    Article  PubMed  PubMed Central  Google Scholar 

  44. Kim Y, Lee YS, Choe J, Lee H, Kim YM, Jeoung D (2008) CD44-epidermal growth factor receptor interaction mediates hyaluronic acid-promoted cell motility by activating protein kinase C signaling involving Akt, Rac1, Phox, reactive oxygen species, focal adhesion kinase, and MMP-2. J Biol Chem 283(33):22513–28. https://doi.org/10.1074/jbc.M708319200

  45. Li Z et al (2017) The OncoPPi network of cancer-focused protein-protein interactions to inform biological insights and therapeutic strategies. Nat Commun 8:14356. https://doi.org/10.1038/ncomms14356

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Chen P et al (2020) Circadian regulator CLOCK recruits immune-suppressive microglia into the GBM tumor microenvironment. Cancer Discov 10(3):371–381. https://doi.org/10.1158/2159-8290.CD-19-0400

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Wang F, Li C, Han F, Chen L, Zhu L (2021) BMAL1 may be involved in angiogenesis and peritumoral cerebral edema of human glioma by regulating VEGF and ANG2. Aging (Albany NY) 13(22):24675–24685. https://doi.org/10.18632/aging.203708

    Article  PubMed  CAS  Google Scholar 

  48. Matias D et al (2018) Microglia/astrocytes-glioblastoma crosstalk: crucial molecular mechanisms and microenvironmental factors. Front Cell Neurosci 12:235. https://doi.org/10.3389/fncel.2018.00235

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. O’Brien ER, Howarth C, Sibson NR (2013) The role of astrocytes in CNS tumors: pre-clinical models and novel imaging approaches. Front Cell Neurosci 7:40. https://doi.org/10.3389/fncel.2013.00040

    Article  PubMed  PubMed Central  Google Scholar 

  50. Escartin C et al (2021) Reactive astrocyte nomenclature, definitions, and future directions. Nat Neurosci 24(3):312–325. https://doi.org/10.1038/s41593-020-00783-4

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Du Z et al (2022) Association of glioma CD44 expression with glial dynamics in the tumour microenvironment and patient prognosis. Comput Struct Biotechnol J 20:5203–5217. https://doi.org/10.1016/j.csbj.2022.09.003

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Maroni MJ et al (2018) Constant light alters serum hormone levels related to thyroid function in male CD-1 mice. Chronobiol Int 35(10):1456–1463. https://doi.org/10.1080/07420528.2018.1488259

    Article  PubMed  CAS  Google Scholar 

  53. Rumanova VS, Okuliarova M, Zeman M (2020) Differential effects of constant light and dim light at night on the circadian control of metabolism and behavior. Int J Mol Sci 21(15):5478. https://doi.org/10.3390/ijms21155478

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Duhart JM, Brocardo L, Caldart CS, Marpegan L, Golombek DA (2017) Circadian alterations in a murine model of hypothalamic glioma. Front Physiol 8:864. https://doi.org/10.3389/fphys.2017.00864

    Article  PubMed  PubMed Central  Google Scholar 

  55. Abdraboh ME et al (2022) Constant light exposure and/or pinealectomy increases susceptibility to trichloroethylene-induced hepatotoxicity and liver cancer in male mice. Environ Sci Pollut Res Int 29(40):60371–60384. https://doi.org/10.1007/s11356-022-19976-4

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  56. Anisimov VN et al (2012) Light-at-night-induced circadian disruption, cancer, and aging. Curr Aging Sci 5(3):170–177. https://doi.org/10.2174/1874609811205030002

    Article  PubMed  Google Scholar 

  57. Khan S et al (2019) Impact of chronically alternating light-dark cycles on circadian clock mediated expression of cancer (glioma)-related genes in the brain. Int J Biol Sci 15(9):1816–1834. https://doi.org/10.7150/ijbs.35520

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  58. Khan S, Yong VW, Xue M (2021) Circadian disruption in mice through chronic jet lag-like conditions modulates molecular profiles of cancer in nucleus accumbens and prefrontal cortex. Carcinogenesis 42(6):864–873. https://doi.org/10.1093/carcin/bgab012

    Article  PubMed  CAS  Google Scholar 

  59. Guerrero-Vargas NN et al (2017) Circadian disruption promotes tumor growth by anabolic host metabolism, experimental evidence in a rat model. BMC Cancer 17(1):625. https://doi.org/10.1186/s12885-017-3636-3

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  60. Ohta H et al (2005) Constant light desynchronizes mammalian clock neurons. Nat Neurosci 8(3):267–269. https://doi.org/10.1038/nn1395

    Article  PubMed  CAS  Google Scholar 

  61. Wagner PM et al (2021) Adjusting the molecular clock: the importance of circadian rhythms in the development of glioblastomas and its intervention as a therapeutic strategy. 22(15):8289. https://doi.org/10.3390/ijms22158289

  62. Cederroth CR et al (2019) Medicine in the fourth dimension. Cell Metab 30(2):238–250. https://doi.org/10.1016/j.cmet.2019.06.019

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

Special thanks to Dr. Santiago Plano (Institute for Biomedical Research (BIOMED), Catholic University of Argentina (UCA), Buenos Aires, Argentina) for the assistance in mice activity recordings.

Funding

This work was supported by grants from Universidad Nacional de Quilmes (PUNQ 1397/16) and by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PICT 2099–2014) from Argentina. Study design, sample collection, data analysis and interpretation, and writing of the manuscript were performed by the authors with no participation of the funding agencies.

Author information

Authors and Affiliations

  1. Laboratorio de Cronobiología, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes/CONICET, Roque S. Peña 352, B1876BXD, Bernal, Buenos Aires, Argentina

    Laura L. Trebucq & Juan J. Chiesa

  2. Laboratorio de Biotransformaciones y Química de Ácidos Nucleicos, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes/CONICET, Roque S. Peña 352, B1876BXD, Bernal, Buenos Aires, Argentina

    Nicolas Salvatore

  3. CIQUIBIC-CONICET y Departamento de Química Biológica Ranwel Caputto, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, X5000HUA, Córdoba, Argentina

    Paula M. Wagner

  4. Laboratorio Interdisciplinario del Tiempo (LITERA), Universidad de San Andrés, B1644BID, Victoria, Buenos Aires, Argentina

    Diego A. Golombek

Authors
  1. Laura L. Trebucq
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nicolas Salvatore
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paula M. Wagner
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Diego A. Golombek
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Juan J. Chiesa
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

LLT performed all the experiments and wrote the manuscript. NS assisted in performing the in vivo experiments. JJC was a major contributor in writing the manuscript and directing the study design and data interpretation. PMW served as technical advisor and wrote the manuscript. DAG served as experimental advisor and wrote the manuscript. All authors have read and approved the manuscript.

Corresponding author

Correspondence to Juan J. Chiesa.

Ethics declarations

Ethics Approval

Protocols with animals were supervised and approved by the Institutional Animal Care and Use Committee of the Universidad Nacional de Quilmes (Protocol resolution 011-15) in accordance with international standards.

Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Trebucq, L.L., Salvatore, N., Wagner, P.M. et al. Circadian Clock Gene bmal1 Acts as a Tumor Suppressor Gene in a Mice Model of Human Glioblastoma. Mol Neurobiol (2024). https://doi.org/10.1007/s12035-023-03895-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s12035-023-03895-7

Keywords

Search

Navigation