The unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes MAP1LC3B and ATG5

Abstract

Tumor hypoxia is a common microenvironmental factor that adversely influences tumor phenotype and treatment response. Cellular adaptation to hypoxia occurs through multiple mechanisms, including activation of the unfolded protein response (UPR). Recent reports have indicated that hypoxia activates a lysosomal degradation pathway known as autophagy, and here we show that the UPR enhances the capacity of hypoxic tumor cells to carry out autophagy, and that this promotes their survival. In several human cancer cell lines, hypoxia increased transcription of the essential autophagy genes microtubule-associated protein 1 light chain 3β (MAP1LC3B) and autophagy-related gene 5 (ATG5) through the transcription factors ATF4 and CHOP, respectively, which are regulated by PKR-like ER kinase (PERK, also known as EIF2AK3). MAP1LC3B and ATG5 are not required for initiation of autophagy but mediate phagophore expansion and autophagosome formation. We observed that transcriptional induction of MAP1LC3B replenished MAP1LC3B protein that was turned over during extensive hypoxia-induced autophagy. Correspondingly, cells deficient in PERK signaling failed to transcriptionally induce MAP1LC3B and became rapidly depleted of MAP1LC3B protein during hypoxia. Consistent with these data, autophagy and MAP1LC3B induction occurred preferentially in hypoxic regions of human tumor xenografts. Furthermore, pharmacological inhibition of autophagy sensitized human tumor cells to hypoxia, reduced the fraction of viable hypoxic tumor cells, and sensitized xenografted human tumors to irradiation. Our data therefore demonstrate that the UPR is an important mediator of the hypoxic tumor microenvironment and that it contributes to resistance to treatment through its ability to facilitate autophagy.

Looking at the metabolic consequences of the colchicine-based in vivo autophagic flux assay

“…[33][34][35][36][37] In this regard, recent studies have identified DDIT3 as a direct regulator of numerous genes involved in the autophagic process. [38][39][40] Here, we show that the increase of transcripts levels of several autophagy-and lysosome-related genes in colchicinetreated fish paralleled that of Ddit3 mRNA supporting these previous studies. However, the increase of lysosomal transcripts was not followed by an increase of the lysosomal function (as evidenced by the activity of CTSD), in line with the demonstrated inhibition of the autophagic flux in our samples and the recently published data showing that activation of lysosomal function depends on autophagosome-lysosome fusion.…”

Overview of macroautophagy regulation in mammalian cells

“…In polyQ72-loaded mammalian cells, the phosphorylation of eIF2α up-regulates the expression of Atg12 [157]. During the unfolded protein response, triggered by hypoxia, the transcription factors ATF4 and CHOP, which are regulated by PERK, increase the expression of Map1lc3b and Atg5, respectively [158]. Glyceraldehyde-3-phosphate dehydrogenase, a multifunctional enzyme known to play a role in glycolysis as well as having other less well-understood roles such as transcriptional coactivation, has also been shown to upregulate the transcription of Atg12 to protect cells against caspase-independent cell death [159].…”

BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism

“…In some scenarios, the induction of autophagy is accompanied by increased mRNA levels of certain autophagy genes such as LC3B, ATG5 or ATG12. 5 However, little is known about the mechanisms that controls ATG protein translation. Indeed, there are opposite opinions about the need of protein translation in starvation-induced autophagy.…”