Here we demonstrate that activation of the ER kinase PERK promotes the survival of luminal MECs detached from the ECM. Two recent studies have shown that upon loss of adhesion, MECs activate survival via autophagy (16
) but also accumulate lethal ROS levels in association with reduced ATP production (40
). We now demonstrate that suspension-induced PERK coordinately contributes to the induction of autophagy, the maintenance of ATP production, and the stimulation of a ROS detoxification response. The last most likely allows for autophagy to proceed and protect cells until adhesion is restored. In a 3D morphogenesis model, the enforced activation of PERK results in the aberrant survival and accumulation of cells in the luminal space, indicating that this pathway must be turned off during morphogenesis for proper luminal clearance. The tissue-specific deletion of PERK in the mouse mammary gland tissue results in reduced autophagy and increased apoptosis, whereas both PERK phosphorylation and autophagy are concomitantly increased in human DCIS. Based on these results, we propose that the proautophagic and antioxidant functions of PERK that operate during normal mammary acinar development are subverted in breast tumor cells to survive oxidative stress and resist anoikis.
Autophagy was found to protect luminal MECs lacking ECM contact in an ATG5- and ATG7-dependent manner (16
), but the precise signals triggering this prosurvival mechanism in ECM-detached cells remain largely unclear (9
). Here we show that PERK is an upstream inducer of detachment-induced autophagy. Interestingly, our data implicate the canonical PERK-eIF2α signaling axis as an important transcriptional regulator of multiple autophagy regulators (ATGs) involved in the early steps of autophagosome formation, including ATG5, ATG6/Beclin-1, and ATG8 expression. Among these, ATG6/Beclin1 was the most responsive ATG gene induced by PERK and its downstream effectors. Because transcription of these ATG genes is likely not sufficient to drive autophagy, it is possible that these core components of the autophagy machinery are also subject to posttranscriptional control downstream of PERK activation. Nonetheless, in addition to the induction of these ATG transcripts, our results also demonstrate that PERK activation (using a control dimerization strategy to activate this ER kinase) is sufficient to promote bona fide autophagic degradation. Notably, PERK may directly or indirectly affect other pathways important in autophagy induction, such as the energy-sensing LKB1-AMPK pathway, which is activated when AMP levels increase and consequently leads to both mTOR inhibition (18
) and autophagy activation (29
). Further examination of this salient issue remains a important topic for future study.
In MCF10A 3D cultures, enforced PERK activation during the late stages of morphogenesis allowed viable MECs devoid of any ECM (i.e., surface bound laminin-5) to persistently occupy the luminal space. This phenotype was dependent on ATF4 and ATG7 and ultimately resulted in increased acinar basal cell numbers, suggesting that surviving MECs reattached to the ECM in the basal layer of cells. Luminal MEC survival was reversed by chloroquine, which blocks fusion of autophagosomes to lysosomes (25
). Thus, drugs that interrupt PERK-induced autophagy might have antitumor effects. In agreement with this, chloroquine was shown to limit the survival of breast cancer cells (15
). We conclude that PERK can protect against anoikis and that oncogenic signals that perpetuate PERK activation might promote unwanted MEC survival.
Based on these results, one can speculate that PERK ensures that a MEC inefficiently attached survives via autophagy until proper adhesion is restored. Importantly, we found that these functions of PERK appear to be operational in vivo
. During lactation, mouse mammary epithelial tissue where PERK was conditionally deleted showed an absence of P-PERK signal that was associated with reduced autophagosomes (e.g., LC3 staining). In further support of this, we found that in normal reduction mammoplasty and benign adjacent human tissues, PERK activation was enhanced and LC3-associated autophagy was increased primarily in luminal cells, once again suggesting an anoikis resistance mechanism. Furthermore, in DCIS lesions, where anoikis resistance is thought to lead to luminal filling (11
), strong PERK phosphorylation was associated with prominent LC3 staining. In agreement with our findings, a recent study demonstrated that autophagy is upregulated in DCIS lesions, but the link to PERK was not delineated (15
In addition to the induction of autophagy, PERK senses the accumulation of ROS in suspended cells and induces an antioxidant response dependent on eIF2α phosphorylation (41
). During ECM detachment, this response includes the induction of the glycine transporter Glyt1 and the x(c)-cystine/glutamate exchanger Slc3a2, which is required to transport cystine that upon reduction is converted into the glutathione (GSH) precursor cysteine (22
). These genes are part of the PERK antioxidant response mediated by ATF4, and GSH is a major reducing agent that prevents ROS-mediated damage (6
). PERK can also maintain cellular redox homeostasis through the activation of Nrf2 (8
), which is a PERK substrate and a central regulator of GSH metabolism (42
). In fact, in ErbB2/Neu-driven tumors, PERK-activated Nrf2 is the primary antioxidant response regulator (4
). Thus, we cannot exclude the possibility that PERK-Nrf2 signaling might contribute to decrease ROS levels in suspended cells. NAC was sufficient to reduce PERK activation and LC3 processing, suggesting that the accumulated ROS and possibly misfolding of proteins leads to PERK activation. The latter is needed to reduce ROS because expression of a PERKΔC or eIF2α Ser51 Ala mutant resulted in increased ROS accumulation and reduced plating efficiency. Further, PERK activation in Fv2E-ΔNPERK-expressing acini completely reversed ROS accumulation. We previously showed that neither PKR nor GCN2 is activated in suspension (41
), reinforcing our findings that this is a PERK-dependent mechanism. Our finding that PERK executes a survival program during mammary acinus morphogenesis in both mouse and human normal tissues and human tumor tissues is significant, as it reveals a potential new role under normal and/or pathophysiological conditions.
PERK has a dual function in mouse mammary epithelium (3
). It can prevent tumorigenesis in aging animals, but it also can aid ErbB2/Neu transformation (5
). The former is in agreement with our previous findings (41
) that if PERK is inhibited using dominant-negative alleles, MECs, but primarily the basal ECM-attached acinar cells, engage in enhanced proliferation and tumor formation. While we had shown that PERK signaling occurred in luminal cells, we had not identified its function at that time (41
). We also found that PERK activation induced p21 and p27 (data not shown), suggesting that its survival function might be tied to a transient growth arrest to promote damage relief. Thus, the growth-suppressive and antioxidant functions of PERK may be necessary to prevent MECs from accumulating damage that can lead to transformation. Importantly, if the transforming signal is an oncogene product like ErbB2, PERK serves as a survival factor allowing tumors to cope with ROS, hypoxia, and ER stress (5
). It is possible that the oncogenic signal is sufficient to overcome PERK-induced cell cycle arrest, leaving the prosurvival function to assist tumorigenesis. We propose that the mechanism of survival normally induced by PERK in luminal cells is hijacked to protect transformed MECs from anoikis.
In suspended cells, a canonical PERK pathway is activated (reference 41
and this study). However, the precise control of PERK activation during ECM detachment remains unclear. One study showed that loss of adhesion of MCF10A cells results in reduced glucose transport, a drop in ATP production, and increased ROS. It is possible that the ER protein-folding machinery, which is ATP dependent, is highly sensitive to the ATP drop and that luminal suspended MECs accumulate misfolded proteins in the ER, resulting in PERK activation as part of a UPR. However, ECM detachment does not appear to induce a canonical UPR, because Grp78, Hsp47, and Erp72/PDI, as well as XBP-1 splicing, is not induced during the same time frame in which PERK activation promotes survival (41
). Although unknown, it may also be possible that suspended MECs, due to altered cell polarity, disrupt vesicular transport, leading to ER client protein accumulation and PERK activation.
In summary, we have found that PERK controls two mechanisms that become simultaneously activated in ECM-detached cells and dictate cell fate: prodeath ROS induction that is antagonized by PERK and a prosurvival autophagy response that is promoted by PERK. We propose that because cell detachment is a critical stress condition, the simultaneous activation of these pathways represents a safeguard mechanism. In the initial hours after detachment, PERK “licenses” suspended cells for survival by inducing autophagy and ROS detoxification, allowing cells to reattach, resolve both autophagy induction and ROS stress, and resume normal functions (G). If MECs do not attach to the ECM, then ROS stress is insurmountable and apoptotic pathways takes over to fully commit these delocalized cells to death via anoikis. We are currently dissecting how transformed mammary epithelial cells hijack these prosurvival functions of PERK during anoikis to provide a robust and persistent anoikis resistance phenotype.