The rapid expansion of tumor cells can result in a microenvironment wherein metabolic nutrients such as glucose and oxygen become limiting. Glucose deprivation or hypoxia leads to perturbation of endoplasmic reticulum (ER) homeostasis, triggering the unfolded protein response pathway (UPR). In normal cells, UPR induction triggers translation suppression, growth arrest and/or apoptosis thereby preventing cellular expansion. However, tumor cells circumvent such limitations and maintain proliferative potential. Understanding how glucose deprivation-induced UPR triggers cell cycle arrest will contribute important mechanistic insights into tumor progression. UPR induction results in the activation of the ER-resident protein kinase PERK, which in turn phosphorylates eIF2α contributing to UPR-triggered translation inhibition and cell cycle arrest. We have explored the possibility that UPR induction regulates ribosome biogenesis and maturation. We have initially focused on a GFP-tagged ribosome subunit protein L5 (GFP-L5) as a probe for ribosome maturation. Following transfection into mouse embryonic fibroblasts (MEFs) derived from wild-type or PERK null mice, subcellular localization of GFP-L5 was visualized by confocal microscopy and intracellular shuttling of GFP-L5 was assessed by Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP). UPR induction by glucose deprivation or tunicamycin treatment triggered the nuclear sequestration of L5 and altered the kinetics of intracellular shuttling of GFP-L5 in PERK-dependent manner. To determine whether PERK-dependent regulation was an indirect consequence of translation inhibition via eIF2α phosphorylation, similar experiments were performed in MEFs harboring a homozygous knock-in of a non-phosphorylatable eIF2α allele (eIF2α A/A). Localization and intracellular shuttling of GFP-L5 was still sensitive to UPR induction in eIF2α A/A cells, suggesting PERK-dependent regulation is independent of eIF2α phosphorylation. GFP-L5 localization and shuttling was insensitive to rapamycin treatment, suggesting the UPR-triggered effect is not mediated through inhibition of mTOR. While mis-localization of ribosomal subunit proteins is likely to contribute to the general decrease in the rate of protein synthesis, perturbation of ribosome biogenesis and maturation is also known to contribute to the activation of the p53 tumor suppressor protein. We found activation of p53 and induction of p53 target genes such as p21 and HDM2 in U2OS cells following glucose deprivation. The mechanism whereby glucose deprivation and subsequent PERK activation contribute to p53 regulation is under investigation. Our preliminary data strongly suggest a role of the UPR in regulating ribosome biogenesis and maturation, which may couple the UPR to cell cycle arrest through HDM2/p53.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]