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Deficiency of ERO1 and activation of protein translation synergistically impair breast tumor resilience

ABSTRACT: Endoplasmic reticulum (ER) stress triggers an adaptive response which fosters tumor cell survival and resilience to stress conditions. Activation of the endoplasmic reticulum stress response, through its PERK branch, promotes the phosphorylation of the α-subunit of translation initiation factor eIF2alpha, thereby repressing general protein translation and selectively augmenting the translation of ATF4 with the downstream CHOP transcription factor and the protein disulfide oxidase ERO1. Here, we show that ISRIB, a small molecule, which inhibits the action of the phosphorylated α-subunit of eIF2, thereby activating protein translation, synergistically interacts with the genetic deficiency of protein disulfide oxidase ERO1 enfeebling tumor growth and spreading. ISRIB represses CHOP signal but surprisingly does not inhibit ERO1. Mechanistically, ISRIB increases the ER protein load with a prominent perturbing effect on ERO1 deficient Triple-Negative breast cells, which have adapted to live with low client protein load, while ERO1 deficiency selectively impairs VEGF-dependent angiogenesis. Strikingly, ERO1-deficient Triple Negative Breast Cancer xenografts have augmented ER stress response and PERK branch. In vivo, ISRIB synergistically with ERO1 deficiency inhibits the growth of Triple-Negative Breast cancer xenografts by impairing proliferation and angiogenesis, while it is not effective on the xenograft counterparts with ERO1. In summary, these results demonstrate that ISRIB together with ERO1 deficiency synergistically shatters a feature of the adaptive ER stress response while ERO1 deficiency selectively impairs angiogenesis in tumors, thereby together promoting tumor cytotoxicity. Therefore, our findings suggest two surprising findings in breast tumors: ERO1 is not regulated via CHOP and ISRIB represents a therapeutic option to efficiently inhibit tumor progression in those tumors with limited ERO1 and high PERK.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Homo sapiens

SUBMITTER: Marco Bolis

PROVIDER: E-MTAB-11313 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Drug-induced liver injury (DILI) is an important clinical problem. Here we used a genomics approach to establish the critical drug-induced toxicity pathways that act in synergy with the pro-inflammatory cytokine tumor necrosis factor (TNF) to cause cell death of liver HepG2 cells. Transcriptomics of the cell injury stress response pathways initiated by two hepatoxicants, diclofenac and carbamazepine, revealed the endoplasmic reticulum (ER) stress/translational initiation signaling and Nrf2 antioxidant signaling as two major affected pathways, which was similar to that observed for the majority of ~80 DILI compounds in primary human hepatocytes. The ER stress was primarily related to PERK and ATF4 activation and subsequent expression of CHOP, which was all independent of TNFM-NM-1 signaling. Identical ATF4 dependent transcriptional programs were observed in primary human hepatocytes as well as primary precision cut human liver slices. Targeted RNA interference studies revealed that while ER stress signaling through IRE1M-NM-1 and ATF6 acted cytoprotective, activation of the ER stress protein kinase PERK and subsequent expression of CHOP was pivotal for the onset of drug/TNF-induced apoptosis. While inhibition of the Nrf2-dependent adaptive oxidative stress response enhanced the drug/TNF cytotoxicity, Nrf2 signaling did not affect CHOP expression. Both hepatotoxic drugs enhanced expression of the translational initiation factor EIF4A1, which was essential for CHOP expression and drug/TNF-mediated cell killing. Our data support a model in which enhanced drug-induced translation initiates PERK-mediated CHOP signaling in an EIF4A1 dependent manner, thereby sensitizing towards caspase-8-dependent TNF induced apoptosis. Liver slices (diameter 4 mm, thickness 250 M-BM-5m) were pre-incubated at 37M-BM-0C for 1h individually in a well containing 1.3 ml WilliamsM-bM-^@M-^Y medium E with glutamax-1 (Gibco, Paisley, UK), supplemented with 25 mM D-glucose and 50 M-BM-5g/ml gentamicin (Gibco, Paisley, UK) (WEGG medium) in a 12-well plate with shaking (90 times/min) under saturated carbogen atmosphere.

Project description:Drug-induced liver injury (DILI) is an important clinical problem. Here we used a genomics approach to establish the critical drug-induced toxicity pathways that act in synergy with the pro-inflammatory cytokine tumor necrosis factor (TNF) to cause cell death of liver HepG2 cells. Transcriptomics of the cell injury stress response pathways initiated by two hepatoxicants, diclofenac and carbamazepine, revealed the endoplasmic reticulum (ER) stress/translational initiation signaling and Nrf2 antioxidant signaling as two major affected pathways, which was similar to that observed for the majority of ~80 DILI compounds in primary human hepatocytes. The ER stress was primarily related to PERK and ATF4 activation and subsequent expression of CHOP, which was all independent of TNFÎ± signaling. Identical ATF4 dependent transcriptional programs were observed in primary human hepatocytes as well as primary precision cut human liver slices. Targeted RNA interference studies revealed that while ER stress signaling through IRE1Î± and ATF6 acted cytoprotective, activation of the ER stress protein kinase PERK and subsequent expression of CHOP was pivotal for the onset of drug/TNF-induced apoptosis. While inhibition of the Nrf2-dependent adaptive oxidative stress response enhanced the drug/TNF cytotoxicity, Nrf2 signaling did not affect CHOP expression. Both hepatotoxic drugs enhanced expression of the translational initiation factor EIF4A1, which was essential for CHOP expression and drug/TNF-mediated cell killing. Our data support a model in which enhanced drug-induced translation initiates PERK-mediated CHOP signaling in an EIF4A1 dependent manner, thereby sensitizing towards caspase-8-dependent TNF induced apoptosis. We sought to determine the effect of co-exposure of certain drugs associated with idiosyncratic DILI with the cytokine TNFalpha to mimic drug exposure with inflammation signaling in HepG2 cells Human hepatoma HepG2 cells were obtained from American Type Culture Collection (ATCC, Wesel, Germany), cultured in DMEM supplemented with 10% (v/v) FBS, 25 U/mL penicillin and 25 Âµg/mL streptomycin and used for experiments between passage 5 and 20.

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Deficiency of ERO1 and activation of protein translation synergistically impair breast tumor resilience

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