Methionine restriction activates the integrated stress response in triple-negative breast cancer cells by a GCN2- and PERK-independent mechanism.

Transformed cells are often selectively susceptible to depletion of the amino acid methionine, which induces growth arrest and/or apoptosis. In non-transformed cells, amino acid deficiency is sensed by two stress-activated kinases, general control nonderepressible 2 (GCN2) and protein kinase R-like endoplasmic reticulum kinase (PERK), which phosphorylate and inactivate elongation initiation factor 2 α (eIF2α), thereby suppressing global mRNA translation and inducing activated transcription factor (ATF4). ATF4 and its downstream transcriptional targets including Sestrin-2 constitute an adaptive integrated stress response. We postulated that methionine depletion activates the integrated stress response in breast cancer cells by a GCN2- and/or PERK-dependent mechanism and that selective disruption of one or both of these kinases would enhance the therapeutic activity of methionine restriction. Here we demonstrate that methionine restriction induces eIF2α phosphorylation and enhances ATF4 gene expression and protein levels of ATF4 and Sestrin-2 in triple (ER/PR/HER2)-negative breast cancer (TNBC) cells. However, knockdown of GCN2, PERK or both in TNBC cells did not prevent induction of ATF4 or Sestrin-2 by methionine restriction. In contrast, deletion of GCN2 in murine embryonic fibroblasts abrogated ATF4 and Sestrin-2 induction in response to methionine restriction. Moreover, knockdown of GCN2, PERK or both did not affect TNBC cell growth or apoptosis in response to methionine restriction. Overall, our findings point to a GCN2- and PERK-independent mechanism(s) by which methionine restriction activates the integrated stress response in TNBC cells. Elucidation of this pathway(s) could lead to strategies to enhance the therapeutic response of methionine restriction.