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 3

ATF4-mediated autophagy provides cytoprotection against anoikis.

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ATF4-mediated autophagy provides cytoprotection against anoikis. (A) Lev...
(A) Levels of autophagy in HT1080 cells grown in attached or suspension conditions were measured by immunoblotting with antibodies against LC3B (LC3BI and LC3BII) and p62. (B) Differences in autophagy induction between shNT and shATF4.HT1080 cells in suspension were similarly measured and compared with cells incubated with media devoid of serum (EBSS). (C) Autophagy flux assay was performed by treating cells with E-64d (10 μg/ml) and 10 μg/ml pepstatin A (Pep) grown in suspension cultures for 48 hours. (D) Immunoblots for LC3B in shNT and shATF4.HT1080 cells transfected with either control (shATF4 + Ad-Cre) or with mATF4 (shATF4 + Ad-mATF4). Ratio of LC3BII to LC3BI is represented as normalized to the attached condition of shNT and shATF4.HT1080 cells. (E) Autophagy was inhibited with increasing concentration of spautin-1, and cell lysates were analyzed by Western blots. (F) HT1080 cells were treated with spautin-1 (5 μM) for indicated time points. Immunoblots for autophagy and apoptosis markers were similarly performed. All immunoblots are representative of 2 independent experiments. (G) Mean cell survival from 3 independent experiments was measured and represented (n = 3, mean ± SD). **P < 0.01, Student’s t test. (H) mRNA levels for ATG5, ATG7, and ULK1 relative to 18S rRNA were measured in shNT and shATF4.HT1080 cells grown in suspension for 48 hours by RT-PCR. Data are represented as mean fold change compared with attached cultures for 3 independent experiments (n = 3, mean ± SD). **P < 0.01, Student’s t test.