Project 1- Micro-RNA-dependent signaling by the UPR

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Abstract Rapidly proliferating cancer cells must thrive in a microenvironment wherein metabolic nutrients such as glucose, oxygen and growth factors become limiting as tumor volume expands beyond the established vascularity of the tissue. In normal cells, limits in nutrient availability trigger growth arrest and/or apoptosis thereby preventing cellular expansion under such conditions. Our scientific premise is that the pro-survival activities of PERK reflect the combined impact of PERK-dependent differential control of protein synthesis and gene expression. In the previous funding cycle, we identified miR-211 and miR-216b as a PERK-ATF4 regulated micro-RNAs that regulate cell survival. We identified three critically important miR-211 targets. The first is the pro-apoptotic transcription factor, Chop. The second target is the Bmal1-Clock heterodimeric transcription factor that functions as the primary driver of circadian gene expression and thus circadian oscillation. Both Bmal1 and Clock are direct targets of miR-211 and are repressed by miR-211 during a UPR. Through analysis of PERK-miR-211 regulation of Bmal1 and the circadian clock, we discovered that 1) circadian gene expression is disrupted by the UPR, 2) Bmal1 loss contributes to PERK-dependent regulation of mRNA translation. We propose a hypothesis wherein the UPR inducible micro-RNAs (miR- 211, miR-217) regulate cell fate by silencing key targets such as Bmal1 which thereby contributes to PERK- dependent translational regulation and cell survival. To address this hypothesis, we propose three specific aims. Aim 1, will defined the critical targets of PERK-miR211-Bmal1 regulation and their contribution to cell survival. Aim 2 will define the role of miR-217 in antagonizing miR-211 regulation of its transcriptional targets. Aim 3 will define the role of miR-211-dependent regulation of Bmal1 to the development and progression of B-cell lymphoma. There are obvious points of cross-talk between this proposal and Project 2 which focuses on how the UPR antagonizes myc induced apoptosis and ATF4-dependent regulation of tumor cell metabolism. Through collaboration with Project 2, we will assess the role of miR-211 and Bmal1 as a mediator of tumor cell metabolism and survival. Through collaboration with Project 3, we will determine how miR-211, miR-217 and Bmal1 regulate IFNAR1 signaling and regulation in cytotic T- lymphocytes. The findings steming from work proposed herein will provide a foundation for the design of novel anti-cancer therpeutics.