The Transcription Factor Atf4 Regulates Resistance To Anoikis And Promotes Metastasis In Fibrosarcoma Via Cooperative Upregulation Of Heme Oxygenase-1 With Nrf2

The Integrated Stress Response (ISR) plays a critical role in cancer cell survival, and targeting the ISR results in inhibition of tumor progression. A critical aspect of ISR involves the preferential translation of activating transcription factor 4(ATF4), a transcriptional factor regulating genes involved in metabolism, nutrient uptake, and anti-oxidant responses. We previously demonstrated that ATF4 expression is significantly increased in tumors compared to corresponding normal tissue, and that ablation of ATF4 compromised primary tumor growth in mice. Based on the central roles that ATF4 target genes play in pro-survival processes, we hypothesized that ATF4 might also play a role in tumor metastasis. Upon loss of matrix attachment, a critical step in the metastatic process, we found induction of phosphorylation of the translation factor eIF2α mediated by upstream kinase PERK in human adenocarcinoma HT1080 cells and colorectal adenocarcinoma DLD1 cells. Increased eIF2α phosphorylation resulted in translational upregulation of ATF4 and its transcriptional targets CHOP, ASNS and ATF3. Interestingly, failure to induce ISR and upregulate ATF4 resulted in increased apoptosis following matrix detachment - a process known as anoikis. Furthermore, we demonstrate that ATF4 promotes anoikis resistance by activating a coordinated program of autophagy and anti-oxidant responses. Upon detachment, ATF4 activates cytoprotective autophagy by transcriptionally upregulating several key autophagic genes (Atg5, Atg7 and Ulk1). Simultaneously, ATF4 also induces the expression of the heme oxygenase 1 (HO-1) - a major antioxidant enzyme. Activation of HO-1 following matrix detachment occurs by coordinated upregulation of ATF4 and the antioxidant PERK dependent transcription factor Nrf2, which converge to bind on antioxidant regulatory elements (ARE) in the HO-1 promoter. Failure to either initiate the autophagy response or HO-1 induction sensitizes cells to anoikis. In agreement with our in vitro observations, HT1080 cells harboring ATF4 shRNA (shATF4) injected through tail vein of nude mice fails to establish metastatic lung colonization after 4 weeks compared to the non-targeting (shNT) counterparts. Immunohistochemical analysis on the tumor bearing lungs show high and colocalized expression of ATF4 and HO-1. Reconstituting either ATF4 or HO-1 expression in ATF4-deficient cells, rescues the tumor lung colonization phenotype. Finally, higher expression of HO-1 and ATF4 was found in human primary as well as metastatic tumors compared to normal epithelium or stromal tissue and correlated with reduced overall survival of lung adenocarcinoma and glioblastoma patients. Collectively, these results establish ATF4 as a major player in tumor metastasis, and the combined activity of its downstream targets HO-1 and Nrf2 as a critical mediator in this process. Citation Format: Souvik Dey, Carly M. Sayers, 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. The transcription factor ATF4 regulates resistance to anoikis and promotes metastasis in fibrosarcoma via cooperative upregulation of Heme Oxygenase-1 with Nrf2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1262. doi:10.1158/1538-7445.AM2015-1262