The integrated stress response effector ATF4: Characterization of a small molecule modulator of its expression and investigation of the role of ATF4 in metastasis

The Integrated Stress Response (ISR) and the Unfolded Protein Response (UPR) are signaling programs that enable cellular adaptation to stressful conditions like hypoxia and nutrient deprivation in the tumor microenvironment. An important effector of these pathways is ATF4, a transcription factor that regulates genes involved in redox homeostasis, amino acid metabolism and transport, autophagy, and angiogenesis. However, prolonged activation of the UPR/ISR and resultant ATF4 expression can be cytotoxic under certain conditions. In these studies, we characterized a small molecule compound, E235, which activated the ISR and dose-dependently increased levels of ATF4 in transformed cells. A dose-dependent decrease in viability was observed in several mouse and human tumor cell lines, and knockdown of ATF4 significantly increased the anti-proliferative effects of E235. Interestingly, low μM doses of E235 induced senescence in many cell types. E235-mediated induction of senescence was not dependent on p21 or p53; however, p21 conferred protection against the growth inhibitory effects of E235. Treatment with E235 resulted in an increase in cells arrested at the G2/M phase. E235 also activated DNA damage response signaling, although E235 did not appear to cause physical DNA damage. Induction of γ-H2AX was abrogated in ATF4 knockdown cells. Together, these results suggest that modulation of the ISR pathway with the small molecule E235 could be a promising anti-tumor strategy. We also investigated the potential role of ATF4 in tumor metastasis. Constitutive knockdown of ATF4 expression in HT1080 fibrosarcoma cells completely prevented the lung colonization of these cells when injected intravenously in mice. This phenomenon may be tumor type specific however, as ATF4 ablation in mouse mammary carcinoma cells had no effect on the ability of these cells to colonize various organs of the mice. In addition, we generated HT1080 cells with an inducible knockdown of ATF4 and demonstrated an increased tumor growth rate upon downregulation of ATF4 expression. However, ATF4 knockdown did not affect the migration of HT1080 cells. This, together with results from a colleague showing that ATF4 protects cells from anoikis, led us to propose that ATF4 may be facilitating metastasis by protecting tumor cells during detachment and circulation