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.

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.