Abstract A45: PERK-Inhibition as a possible therapy for hypoxia-induced solitary dormant tumor cells
Authors: Sandoval Miguel Vizarreta , Fluegen Georg , Staschke Kirk A. , Calvo-Vidal Veronica , Aguirre-Ghiso Julio A. 
Published: May 2019

Abstract
Disseminated tumor cells (DTCs) travel to secondary organs, where they may remain viable for extended amounts of time (years to decades). This state, known as cellular dormancy, is defined as a state of quiescence and cells remain solitary. Preliminary data from our group strongly suggests that single DTCs originating from hypoxic microenvironments within the primary tumor may be more prone to enter a prolonged cellular dormancy at secondary sites. Hypoxic stress also leads to the activation of the endoplasmic reticulum (ER) stress sensor PKR-like ER Kinase (PERK), which promotes the activation of survival genes through the unfolded protein response (UPR). We hypothesize that hypoxia-induced dormant cells require PERK for survival and that through PERK inhibition we can target dormancy. Here, we evaluate PERK inhibition as a way to selectively eliminate quiescent head and neck squamous cell carcinoma (HNSCC) tumor cells. To determine whether hypoxia induces dormancy and PERK activation we cultured HNSCC tumorigenic HEp3 (T-HEp3) cells in normoxic (21% O2) and hypoxic (1% O2) conditions for 8 and 24 hours, and measured the expression of quiescence markers (p27), dormancy markers (DEC2 & NR2F1), and PERK activation (ATF4) via western blot. We found that only at 8 hours p27, DEC2 and NR2F1 are higher expressed in HNSCC in hypoxic conditions in comparison to normoxic conditions. NR2F1 and DEC2 have been shown to promote dormancy by limiting proliferation via p27 induction and mediating the expression of pluripotency genes. After 8 and 24 hours, ATF4, a downstream target of PERK, was upregulated in hypoxic over normoxic cells. These findings support that hypoxic cells express dormancy markers simultaneously with active PERK signaling. To test HNSCC cell sensitivity to PERK inhibition, we seeded T-HEp3-TET-On-H2B-GFP cells at single cell level in a 3D Matrigel. These cells express a green fluorescent protein (GFP) in the presence of doxycycline (Dox). By seeding at a single cell level the solitary cells enter a quiescent phenotype. H2B-GFP was induced with Dox in 2D tissue culture for 3 days, prior to seeding in the Matrigel. Cells were then cultured in Dox free Matrigel in normoxic conditions for 10 days, daily treatment with PERK inhibitor LY4 was started at day 4. As a marker for quiescence, we monitored over time the retention of the H2B-GFP label in the nucleosomes. Treatment with 2μM and 6μM LY4 led to a significant decrease (p = 0.03, 0.0003, respectively) in the percentage of green label retaining cells at day 10 and 7, respectively. The total number of live cells did not increase over time (p = 0.17), arguing that the loss of H2B-GFP+ cells was not due to an increase in proliferation and dilution of the label but rather due to cell death of the quiescent, GFP positive cells. Further experiments will confirm the effect of such treatment on the hypoxic cells and the rate of death. These results indicate that dormant cells, either induced through hypoxic conditioning or single cell state, upregulate a set of dormancy markers as well as PERK. Quiescent cells seem to be dependent on PERK activity for their survival. The PERK inhibition selectively affected the survival of the quiescent, dormant-like H2B-GFP positive cells. This initial data suggests that PERK might represent a novel therapeutic target against disseminated DTCs, for which no therapeutic strategy is currently available.
Citation Format: Miguel Vizarreta Sandoval, Georg Fluegen, Kirk A. Staschke, Veronica Calvo-Vidal, Julio A. Aguirre-Ghiso. PERK-Inhibition as a possible therapy for hypoxia-induced solitary dormant tumor cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A45.