3D Culture Models with CRISPR Screens Reveal Hyperactive NRF2 as a Prerequisite for Spheroid Formation via Regulation of Proliferation and Ferroptosis

Mol Cell. 2020 Dec 3;80(5):828-844.e6. doi: 10.1016/j.molcel.2020.10.010. Epub 2020 Oct 30.

Abstract

Cancer-associated mutations that stabilize NRF2, an oxidant defense transcription factor, are predicted to promote tumor development. Here, utilizing 3D cancer spheroid models coupled with CRISPR-Cas9 screens, we investigate the molecular pathogenesis mediated by NRF2 hyperactivation. NRF2 hyperactivation was necessary for proliferation and survival in lung tumor spheroids. Antioxidant treatment rescued survival but not proliferation, suggesting the presence of distinct mechanisms. CRISPR screens revealed that spheroids are differentially dependent on the mammalian target of rapamycin (mTOR) for proliferation and the lipid peroxidase GPX4 for protection from ferroptosis of inner, matrix-deprived cells. Ferroptosis inhibitors blocked death from NRF2 downregulation, demonstrating a critical role of NRF2 in protecting matrix-deprived cells from ferroptosis. Interestingly, proteomics analyses show global enrichment of selenoproteins, including GPX4, by NRF2 downregulation, and targeting NRF2 and GPX4 killed spheroids overall. These results illustrate the value of spheroid culture in revealing environmental or spatial differential dependencies on NRF2 and reveal exploitable vulnerabilities of NRF2-hyperactivated tumors.

Keywords: oxidative stress, cancer, cell death, ferroptosis, 3D culture, NRF2, CRISPR screening, selenoprotein, GPX4.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • A549 Cells
  • CRISPR-Cas Systems*
  • Cell Culture Techniques*
  • Cell Proliferation*
  • Ferroptosis*
  • Humans
  • NF-E2-Related Factor 2 / genetics
  • NF-E2-Related Factor 2 / metabolism*
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / metabolism*
  • Neoplasms / genetics
  • Neoplasms / metabolism*
  • Neoplasms / pathology
  • Phospholipid Hydroperoxide Glutathione Peroxidase / genetics
  • Phospholipid Hydroperoxide Glutathione Peroxidase / metabolism
  • Spheroids, Cellular / metabolism*
  • Spheroids, Cellular / pathology
  • TOR Serine-Threonine Kinases / genetics
  • TOR Serine-Threonine Kinases / metabolism

Substances

  • NF-E2-Related Factor 2
  • NFE2L2 protein, human
  • Neoplasm Proteins
  • Phospholipid Hydroperoxide Glutathione Peroxidase
  • MTOR protein, human
  • TOR Serine-Threonine Kinases