Endoplasmic reticulum (ER) stress leads to activation of the unfolded protein response (UPR) that results in transient suppression of protein translation to allow recovery but leads to cell death when stress cannot be resolved. Central to initiation of the UPR is the activation of the ER transmembrane kinase protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK). Here we report that the thiol oxidoreductase ERp57 and protein disulfide isomerase-A1 (PDI), which belong to the same family of luminal ER oxidoreductases, have strikingly opposing roles in the regulation of PERK function. In HCT116 colon carcinoma cells, lentiviral depletion of ERp57 resulted in oxidation of PDI and activation of PERK, whereas depletion or chemical inhibition of PDI reduced PERK signaling and sensitized the cancer cells to hypoxia and ER stress. We conclude that oxidized PDI acts as a PERK activator, whereas ERp57 keeps PDI in a reduced state in the absence of ER stress. Thus, our study defines a new interface between metabolic redox signaling and PERK-dependent activation of the UPR and has the potential to influence future cancer therapies that target PERK signaling.

The disulfide isomerase ERp57, originally found in the endoplasmic reticulum, is located in multiple cellular compartments, participates in diverse cell functions and interacts with a huge network of binding partners. It was recently suggested as an attractive new target for cancer therapy due to its critical role in tumor cell proliferation. Since a major bottleneck in cancer treatment is the occurrence of hypoxic areas in solid tumors, the role of ERp57 in cell growth was tested under oxygen depletion in the colorectal cancer cell line HCT116. We observed a severe growth inhibition when ERp57 was knocked down in hypoxia (1% O2) as a consequence of downregulated c-Myc, PLK1, PDPK1 (PDK1) and AKT (PKB). Further, irradiation experiments revealed also a radiosensitizing effect of ERp57 depletion under oxygen deprivation. Compared to ERp57, we do not favour PDPK1 as a suitable pharmaceutical target as its efficient knockdown/chemical inhibition did not show an inhibitory effect on proliferation.

IntroductionFast-growing tumour cells show enhanced protein synthesis and therefore depend on efficient folding for nascent export proteins in the endoplasmic reticulum (ER). Herein, the two most prominent ER resident thiol oxidoreductases ERp57 and PDI play important parts in formation of disulfide bonds in client proteins. This and the finding that both proteins fulfil various roles also in other compartments (i.e. cytoplasm, nucleus, cell membrane) encouraged us to investigate the impact of their depletion on colorectal cancer cells.Material and methodsUsing an inducible knockdown (KD) system we tested ERp57 and PDI deficiency in long term survival assays in normoxia and hypoxia combined with irradiation.Results and discussionsKD of ERp57 or PDI triggered a severe attenuation of proliferation, but only ERp57 deficiency led to activation of the PERK-dependent UPR and apoptosis. When combined with an ERp57 KD, irradiation displayed the most dramatic growth reduction even under 1% oxygen. The absence of ERp57 reduced expression of cellular proliferation factors like c-Myc, PLK-1, AKT, PDPK1, ERK1,2 and others. Further, we demonstrated for the first time that PDI is an essential activator of the ER stress sensor PERK that enforces cancer cell survival under global ER stress in hypoxia. In the absense of ER stress, ERp57 functions as a reductase for PDI that keeps PERK in an inactive state.ConclusionOur data identified ERp57 and PDI as promising new targets for a mono- and combination anti-cancer therapy due to multiple cellular points of attack.