In response to diverse environmental and physiological stresses, eukaryotic cells activate a homeostatic program of gene expression known as Integrated Stress Response (ISR). Activation of ISR involves phosphorylation of eukaryotic translation initiation factor eIF2α (eIF2α-P) repressing global translation along with preferential translation of activating transcription factor 4(ATF4), a transcriptional regulator for an array of genes involved in metabolism, nutrient uptake, and anti-oxidant responses. We previously demonstrated that ATF4 expression is significantly increased in tumors as compared to the normal tissue and ablation of ATF4 expression inhibits tumor growth in mice. Recent results from our group also suggest a role of ATF4 in metastasis. Extracellular matrix detachment causes cell death in normal cells by anoikis- an apoptosis mechanism that is known to be deregulated in highly aggressive metastatic tumor cells. Here we report a possible role of ATF4 in rendering resistance towards anoikis mediated cell death. Human adenocarcinoma HT1080 cells harboring ATF4 shRNA injected through tail vein of nude mice fails to establish metastatic lung colonization after 4 weeks as compared to the wild type counterparts. Using suspension cultures we show that detachment of HT1080 cells from extracellular matrix induces ISR via phosphorylation of eIF2α phosphorylation and activation of ATF4. Down regulating ATF4 expression either through shRNA (shATF4) mediated knock down or blocking eIF2α kinase PERK significantly reduced cell survival with increased apoptosis. Inducible knockdown of ATF4 with doxycycline blocked ATF4 expression in suspension and rendered cells sensitive to anoikis induced cell death. Survival following matrix detachment has been shown to be induced through activation of autophagy - a tightly regulated cellular self-digestive process. We observed a similar induction of autophagy in HT1080 fibrosarcoma cells when subjected to anoikis characterized by increased expression of LC3II and degradation of p62. Blocking the induction of autophagy by spautin-1 renders cells sensitive to anoikis suggesting a pro-survival function. Interestingly, ablation of ATF4 expression fails to sustain the autophagic response which explains the sensitivity of these cells towards anoikis mediated cellular apoptosis. Systemic knockdown of several key factors of autophagic process revealed that Atg5 and ULK1 plays in important role in anoikis mediated autophagy. Loss of both Atg5 and ULK1- both transcriptional targets of ATF4, blocked autophagy and significantly increased apoptosis of HT1080 cells. Collectively, our findings divulge a novel role of ATF4 in providing cancer cells protection against physiologically induced autophagy and thus can be exploited as novel target for aggressive metastatic tumors.

Citation Format: Souvik Dey, Carly M. Sayers, Constantinos Koumenis. Activation of the Integrated Stress Response target ATF4 provides resistance towards anoikis- mediated cell death and promotes metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2934. doi:10.1158/1538-7445.AM2013-2934