Hypoxia is a common feature of solid tumors and a major limiting factor in successful cancer treatment (7
). Here we reveal what we believe is a novel and important mode of autophagy regulation by hypoxia, mediated by transcriptional activation of MAP1LC3B
. We also demonstrate that MAP1LC3B
are direct transcriptional targets of the PERK-dependent transcription factors ATF4 and CHOP. Cells with defective PERK signaling failed to induce MAP1LC3B during hypoxia and lost MAP1LC3B protein during hypoxic exposure. More importantly, PERK/ATF4 regulation of MAP1LC3B was a critical regulator of autophagy and hypoxic cell survival in tumors. We found that in 2 different cell line xenografts and 12 head and neck tumor xenografts, MAP1LC3B and autophagy was strongly activated in hypoxic tumor areas. Blockade of UPR signaling or autophagy through genetic and pharmacological methods reduced hypoxia tolerance in vitro, reduced the levels of viable hypoxic cells in tumor xenografts, and sensitized tumors to irradiation. Treatment with inhibitors of autophagy also selectively reduced cell proliferation during mild hypoxia both in vitro and in tumor xenografts. Taken together, our findings indicate that activation of PERK increases hypoxia tolerance and the resistance of tumors to treatment with irradiation by increasing the capacity of hypoxic cells to carry out autophagy. Consequently, targeting PERK signaling or autophagy may be an effective way to eliminate treatment-resistant, hypoxic tumor cells. Interestingly, previous published data have already demonstrated the potential beneficial effects of targeting autophagy in combination with other standard treatments (49
It is not yet clear how autophagy exerts a protective effect on cell proliferation and survival during hypoxia. During conditions of stress and starvation, cellular organelles and cytoplasmic content are degraded, thereby enabling the cells to recycle amino acids and nutrients to maintain protein synthesis and ATP generation (24
). Since energy homeostasis is compromised during hypoxia, autophagy may function as an important source of metabolic requirements to sustain cell survival. Survival of cells after metabolic stress induced by glucose or amino acid deprivation are similarly dependent on autophagy (51
). However, autophagy also functions as a cytoplasmic quality control mechanism to eliminate protein aggregates and damaged organelles (24
). Defects in autophagy cause accumulation of cytoplasmic inclusion bodies and protein aggregates in the cytoplasm, causing toxicity (54
). During carcinogenesis autophagy suppresses tumor progression through stabilization of the genome (58
), perhaps related to the inability to clear the cell of damaged organelles or toxic protein aggregates. Our results, which link PERK signaling to autophagy through regulation of MAP1LC3B and ATG5, are interesting in this regard since accumulation of unfolded or misfolded proteins in the ER acts as the initial signal for UPR activation. Thus, PERK may stimulate autophagy during hypoxia to help clear the ER of toxic protein aggregates that are responsible for its activation. Several ER stress–inducing agents have previously been shown to induce autophagy in both yeast and mammalian cells. Additionally, both the PERK/eIF2α (60
) and IRE-1 (63
) arms of the UPR have been implicated in autophagy regulation. In yeast, which lacks both PERK and ATF6, IRE-1 promotes an ER-selective form of autophagy that mediates removal of the ER and misfolded protein aggregates. In mammalian cells, PERK signaling is required for autophagy following expression of the expanded polyglutamine 72 repeat (60
) and following exposure to sorafenib and vorinostat (61
). Similar to what we report for hypoxia, PERK-dependent stimulation of autophagy is protective in these examples.
Although previous studies have implicated UPR signaling in autophagy, no direct links between the UPR transcriptional program and autophagy have been reported during hypoxia. Recently it was shown that lack of ATF4 decreased MAP1LC3B expression in bortezemib-treated cells (43
To our knowledge, our study provides the first direct link implicating the PERK-dependent transcription factors ATF4 and CHOP in the transcriptional activation of MAP1LC3B
during hypoxia. MAP1LC3B and ATG5 each play a central role in the 2 ubiquitin-like conjugation systems involved in the formation of autophagosomes (65
). During autophagy, the fraction of MAP1LC3B at the inner membrane of the autophagosome is subject to degradation upon fusion with a lysosome, which must be replenished to allow the cell to maintain autophagy (29
). Our data suggest that this role is fulfilled by the PERK/eIF2α/ATF4 arm of the UPR during hypoxia, and we speculate that this allows cells to survive during prolonged periods of hypoxia. The only other known transcriptional regulator of MAP1LC3B is FoxO3. In skeletal muscles, FoxO3 has been reported to play a similar role in replenishing MAP1LC3B and protecting against muscle atrophy (66
). Interestingly, FoxO3 activity is increased after hypoxic exposure (68
) and thus is an additional potential mediator of autophagy. However, this was not responsible for increased MAP1LC3B during hypoxia, since cells lacking FoxO3 showed normal induction of MAP1LC3B (Supplemental Figure 11).
ATG5 becomes modified on an internal lysine with the ubiquitin-like protein ATG12 as part of the second conjugation system that promotes autophagy. The ATG12-ATG5 conjugate is involved in the initial steps of autophagosome formation (69
), and loss of ATG5 effectively inhibits autophagy. Interestingly, the ATG12-ATG5 complex is also important for processing and lipidation of ATG8/MAP1LC3B, which suggests that the 2 conjugation systems work in synergy to promote the development of the autophagosome (70
). The consequences of hypoxic induction of ATG5 during hypoxia are not clear but may promote activation of autophagy or support an increase in autophagic flux. Understanding the role of ATG5 is further complicated by the fact that it also can promote apoptosis (71
). Overexpression of ATG5 sensitizes to apoptosis upon stimulation with death signals through physically interacting with Bcl-xL and the Fas-associated death domain (FADD) (reviewed in ref. 71
). The involvement of ATG5 in both pro-apoptotic and autophagy pathways suggests it may be instrumental in deciding between autophagy and apoptosis. Interestingly, cells derived from ATG5-deficient MEFs are somewhat more resistant to hypoxia-induced cell death and produce tumors that grow more quickly than their WT counterparts (33
). It is not clear whether these differences arise due to the effects of ATG5 on apoptosis or autophagy, or whether they are MEF dependent. In agreement with our data, Bellot et al. (72
) showed that disruption of autophagy sensitized cells to hypoxia. Here we show that ATG5 is a direct target of CHOP, which, besides autophagy (as shown here), has also been shown to regulate apoptosis (73
). It is therefore tempting to speculate that the connection between CHOP and apoptosis is related to the ability of CHOP to influence the balance between autophagy and apoptosis, but this requires further research.
Our results demonstrate a new mechanism linking the PERK arm of the UPR to hypoxia-induced autophagy. They expand on previous reports demonstrating activation of autophagy by hypoxia through mechanisms involving AMPK (33
) and the HIF1 target gene, BNIP3
). In the conditions used in our study, hypoxia-induced autophagy occurred at least in part independently of BNIP3, since BNIP3 was not expressed in either HT29 or HCT116 cells. Recent evidence showed that removal of BNIP3 is insufficient to block autophagy, but simultaneous removal of BNIP3 and BNIP3L did block autophagy (72
). Additionally, BNIP3 may regulate a more specific form of “mitochondrial autophagy which is important for reducing mitochondrial mass, respiration to prevent formation of toxic reactive oxygen species” (25
). We suggest that PERK- and ATF4-dependent regulation of MAP1LC3B
acts to support autophagy by replenishing MAP1LC3B protein levels and thus the capacity to carry out autophagy during extended periods of hypoxic stress. Consequently, this pathway may act in concert with these other mechanisms that activate or initiate the autophagic process during hypoxia (Figure ). Importantly, this supportive pathway appears to mediate hypoxia tolerance and levels of viable hypoxia in vivo. Targeting this pathway is able to reduce the hypoxic fraction and sensitize tumors to treatment and is thus an attractive therapeutic option to pursue clinically.
Model for recycling and regeneration of MAP1LC3B during hypoxia.