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 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 2

ATF4 confers resistance to anoikis.

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ATF4 confers resistance to anoikis. shNT and shATF4.HT1080 (A) as well a...
shNT and shATF4.HT1080 (A) as well as shNT and shATF4.DLD1 cells (B) were grown in attached or in suspension conditions, and Western blot analysis for markers of the ISR and apoptosis was performed. (C) Mean percentage cell viability from 3 independent experiments was measured in shNT and shATF4.HT1080 cells grown in suspension (n = 3, mean ± SD). *P < 0.05; **P < 0.01, Student’s t test. (D) HT1080 cells stably expressing tetracycline i-shATF4 or i-shNT were grown in attached or suspension conditions for 48 hours or treated with 0.5 μM TG after pretreatment either with DMSO (–) or Dox (+) to alter ATF4 expression. Levels of c-PARP and cleaved caspase-3 were measured and compared with ST treatment. (E) Immunoblots of lysates from shATF4.HT1080 infected with empty vector, control (Ad-Cre), or mATF4 (Ad-mATF4) and grown in attached or suspension cultures for 48 hours. (F) Cell viability by Trypan blue exclusion assay. Percentage of cell survival is represented as mean ± SD from 3 independent experiments (n = 3, mean ± SD). *P < 0.05; **P < 0.01, Student’s t test. All immunoblots are representative of 2 independent experiments.