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.

Rapidly growing tumor cells must synthesize proteins at a high rate and therefore depend on an efficient folding and quality control system for nascent secretory proteins in the endoplasmic reticulum (ER). The ER resident thiol oxidoreductase ERp57 plays an important role in disulfide bond formation. Lentiviral, doxycycline-inducible ERp57 knockdown was combined with irradiation and treatment with chemotherapeutic agents. The knockdown of ERp57 significantly enhanced the apoptotic response to anticancer treatment in HCT116 colon cancer cells via a p53-dependent mechanism. Instead of a direct interaction with p53, depletion of ERp57 induced cell death via a selective activation of the PERK branch of the Unfolded Protein Response (UPR). In contrast, apoptosis was reduced in MDA-MB-231 breast cancer cells harboring mutant p53. Nevertheless, we observed a strong reduction of proliferation in response to ERp57 knockdown in both cell lines regardless of the p53 status. Depletion of ERp57 reduced the phosphorylation activity of the mTOR-complex1 (mTORC1) as demonstrated by reduction of p70S6K phosphorylation. Our data demonstrate that ERp57 is a promising target for anticancer therapy due to synergistic p53-dependent induction of apoptosis and p53-independent inhibition of proliferation.

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Hypoxia-induced resistance of tumor cells to therapeutic treatment is an unresolved limitation due to poor vascular accessibility and protective cell adaptations provided by a network, including PERK, NRF2, and HIF signaling. All three pathways have been shown to influence each other, but a detailed picture remains elusive. To explore this crosstalk in the context of tumor therapy, we generated human cancer cell lines of pancreatic and lung origin carrying an inducible shRNA against NRF2 and PERK. We report that PERK-related phosphorylation of NRF2 is only critical in Keap1 wildtype cells to escape its degradation, but shows no direct effect on nuclear import or transcriptional activity of NRF2. We could further show that NRF2 is paramount for proliferation, ROS elimination, and radioprotection under constant hypoxia (1% O2), but is dispensable under normoxic conditions or after reoxygenation. Depletion of NRF2 does not affect apoptosis, cell cycle progression and proliferation factors AKT and c-Myc, but eliminates cellular HIF-1α signaling. Co-IP experiments revealed a protein interaction between NRF2 and HIF-1α and strongly suggest NRF2 as one of the cellular key factor for the HIF pathway. Together these data provide new insights on the complex role of the PERK-NRF2-HIF-axis for cancer growth.

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.