The Perk-eIF2α -ATF4 arm of the Unfolded Protein Response (UPR) contributes to tumor progression and is a target for therapeutic intervention.

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 4579 Hypoxia/anoxia in the tumor microenvironment promotes tumor aggressiveness and negatively impacts response to therapy. We previously demonstrated that tumor cells under hypoxia activate the endoplasmic reticulum (ER) kinase Perk, thereby inducing phosphorylation of the translation initiation factor eIF2α and translational upregulation of the transcription factor ATF4. The Perk-eIF2α-ATF4 pathway constitutes one component of the Unfolded Protein Response (UPR), a coordinated cellular program that promotes survival under conditions of ER stress. Inactivation of Perk or eIF2α phosphorylation or expression of a dominant-negative Perk in transformed cells, result in significant inhibition of tumor growth due to higher levels of apoptosis inn hypoxic areas and defective angiogenesis compared to tumors with an intact UPR. Notably, we and others found expression of ATF4 in hypoxic areas of clinical samples of human tumors. To gain further insight into the role of ATF4 in tumor progression, we performed microarray gene expression analysis on ATF4+/+ and ATF4-/- Mouse Embryonic Fibroblasts (MEFs), exposed to anoxia or normoxia for 10h. We have identified a number of genes whose expression was significantly changed in both an ATF4-dependent and hypoxia-dependent manner. The mRNA levels of TRB3, a protein induced under nutrient deprivation and of Pyruvate dehydrogenase kinase 1 (PDK1), which inhibits mitochondrial respiration to protect cells in low-oxygen conditions, were induced by anoxia to significantly higher levels in ATF4+/+ compared to ATF4-/- MEFs (22-fold vs. 4-fold for TRB3 and 17-fold vs. 4-fold for PDK1). We are currently investigating the role of ATF4 and its targets in hypoxia tolerance and in tumor progression by knock-down experiments. Interestingly, using MTT and clonogenic survival assays, we also found that hypoxic HeLa and PC-3 cells were significantly more sensitive to the known ER stressors thapsigargin and the FDA-approved proteasome-inhibitor PS-341, compared to normoxic tumor cells. This increased cytotoxicity correlated with increased levels of the UPR effectors ATF4 and Chop, suggesting treatment of hypoxic tumor cells with ER stress-inducing agents produces levels of ER stress that are not compatible with cell survival. More importantly, xenograft tumors from HeLa cells treated with PS-341 showed increased levels of apoptosis in hypoxic areas compared to tumors treated with vehicle alone. Taken together, our results and those of other groups indicate that the UPR is activated by the tumor microenvironment and contributes to tumor progression; however, UPR activation may also provide a unique opportunity to selectively target hypoxic cells with ER stress-inducing agents. This work was supported by NCI grant CA-94214 to CK.