ATF4-dependent induction of heme oxygenase 1 prevents anoikis and promotes metastasis
Souvik Dey, … , J. Alan Diehl, Constantinos Koumenis
Souvik Dey, … , J. Alan Diehl, Constantinos Koumenis
Published July 1, 2015; First published May 26, 2015
Citation Information: J Clin Invest. 2015;125(7):2592-2608. https://doi.org/10.1172/JCI78031.
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Research Article Oncology

ATF4-dependent induction of heme oxygenase 1 prevents anoikis and promotes metastasis

Abstract

The integrated stress response (ISR) is a critical mediator of cancer cell survival, and targeting the ISR inhibits tumor progression. Here, we have shown that activating transcription factor 4 (ATF4), a master transcriptional effector of the ISR, protects transformed cells against anoikis — a specialized form of apoptosis — following matrix detachment and also contributes to tumor metastatic properties. Upon loss of attachment, ATF4 activated a coordinated program of cytoprotective autophagy and antioxidant responses, including induced expression of the major antioxidant enzyme heme oxygenase 1 (HO-1). HO-1 upregulation was the result of simultaneous activation of ATF4 and the transcription factor NRF2, which converged on the HO1 promoter. Increased levels of HO-1 ameliorated oxidative stress and cell death. ATF4-deficient human fibrosarcoma cells were unable to colonize the lungs in a murine model, and reconstitution of ATF4 or HO-1 expression in ATF4-deficient cells blocked anoikis and rescued tumor lung colonization. HO-1 expression was higher in human primary and metastatic tumors compared with noncancerous tissue. Moreover, HO-1 expression correlated with reduced overall survival of patients with lung adenocarcinoma and glioblastoma. These results establish HO-1 as a mediator of ATF4-dependent anoikis resistance and tumor metastasis and suggest ATF4 and HO-1 as potential targets for therapeutic intervention in solid tumors.

Authors

Souvik Dey, Carly M. Sayers, Ioannis I. Verginadis, Stacey L. Lehman, Yi Cheng, George J. Cerniglia, Stephen W. Tuttle, Michael D. Feldman, Paul J.L. Zhang, Serge Y. Fuchs, J. Alan Diehl, Constantinos Koumenis

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Figure 1

Induction of the ISR is critical for resistance to anoikis.

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Induction of the ISR is critical for resistance to anoikis. (A) HT1080 c...
(A) HT1080 cells were grown in attached (ATT) or suspension (SUSP) conditions and treated with 0.5 μM TG or with 1 μM ST. Whole-cell lysates were used to detect levels of p-eIF2α, eIF2α, ATF4, CHOP, c-PARP, cleaved caspase-3 (c-Casp3), and β-actin. (B) Transcript levels of CHOP, ATF3, and ASNS relative to 18S rRNA. Data are represented as mean fold change compared with attached cultures for 3 independent experiments (n = 3, mean ± SD). *P < 0.05; **P < 0.01, Student’s t test. (C) HT080 cells transfected with shNT or shPERK were cultured in attached or suspension conditions, and Western blot analysis was performed. (D) shNT.HT1080 cells were treated with 1 μM PERK inhibitor GSK2606414 (GSK414) in attached or in suspension culture. Immunoblot analysis for the indicated proteins was performed. (E) Cell viability was analyzed by Trypan blue exclusion assay and is represented as the mean percentage cell survival of 3 independent experiments (n = 3, mean ± SD). *P < 0.01; **P < 0.001, by Student’s t test. (F) HT1080 stably transfected with shRNA against GCN2 (shGCN2) and nontargeting counterpart (shNT) were cultured in attached and suspension conditions and immunoblotted for the indicated proteins. All immunoblots are representative of 2 independent experiments.