Critical roles for nitric oxide and ERK in the completion of prosurvival autophagy in 4OHTAM-treated estrogen receptor-positive breast cancer cells

Cancer Lett. 2014 Oct 28;353(2):290-300. doi: 10.1016/j.canlet.2014.07.031. Epub 2014 Jul 25.


Autophagy is a mechanism of tamoxifen (TAM) resistance in ER-positive (ER+) breast cancer cells. In this study, we showed in ER+ MCF7 cells that 4-hydroxytamoxifen (4OHTAM) induced cellular nitric oxide (NO) that negatively regulates cellular superoxide (O2-) and cytotoxicity. 4OHTAM stimulated LC3 lipidation and formation of monodansylcadaverine (MDC)-labeled autophagic vesicles dependent on O2-. Depletion of NO increased O2- and LC3 lipidation, yet reduced formation of MDC-labeled autophagic vesicles. Instead, NO-depleted cells formed remarkably large vacuoles with rims decorated by LC3. The vacuoles were not labeled by MDC or the acidic lysosome-specific fluorescence dye acridine orange (AO). The vacuoles were increased by the late stage autophagy inhibitor chloroquine, which also increased LC3 lipidation. These results suggest NO is required for proper autophagic vesicle formation or maturation at a step after LC3 lipidation. In addition, 4OHTAM induced O2--dependent activation of ERK, inhibition of which destabilized lysosomes/autolysosomes upon 4OHTAM treatment and together with depletion of NO led to necrotic cell death. These results suggest an essential role for endogenous NO and ERK activation in the completion of pro-survival autophagy.

Keywords: Autophagy; Breast cancer; Estrogen receptor; Nitric oxide; Reactive oxygen species; Tamoxifen.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antineoplastic Agents / pharmacology*
  • Autophagy
  • Breast Neoplasms
  • Extracellular Signal-Regulated MAP Kinases / physiology*
  • Female
  • Humans
  • MAP Kinase Signaling System
  • MCF-7 Cells
  • Microtubule-Associated Proteins / metabolism
  • Nitric Oxide / physiology*
  • Nitric Oxide Synthase Type II / metabolism
  • Nitric Oxide Synthase Type III / metabolism
  • Oxygen / metabolism
  • Phagosomes / metabolism
  • Protein Processing, Post-Translational
  • Receptors, Estrogen / metabolism*
  • Tamoxifen / analogs & derivatives*
  • Tamoxifen / pharmacology


  • Antineoplastic Agents
  • MAP1LC3A protein, human
  • Microtubule-Associated Proteins
  • Receptors, Estrogen
  • Tamoxifen
  • afimoxifene
  • Nitric Oxide
  • NOS2 protein, human
  • NOS3 protein, human
  • Nitric Oxide Synthase Type II
  • Nitric Oxide Synthase Type III
  • Extracellular Signal-Regulated MAP Kinases
  • Oxygen