THE TRANSLATIONAL REGULATION OF PRO-APOPTOTIC GENES PROJECT SUMMARY Inactivation of the Retinoblastoma 1 protein (pRB) is one of the most common alterations in cancer. This is because pRB inhibits the activity of three E2F transcription factors, E2F1-3, that control the expression of numerous critical RB-E2F target genes that are required for cancer cell growth, including cell cycle and metabolic factors. In addition to tumor-promoting processes, E2F1-3 also regulates the transcription of tumor-inhibiting genes that play a central role in apoptosis and necrosis. This regulatory circuit links the expression of RB-E2F proliferation and cell death genes, ultimately preventing uncontrolled cell growth. The widespread loss of pRB in cancer underscores a significant puzzle in the field: why don?t pRB-deficient cells simply die? To understand the regulation of the RB-E2F target genes, we profiled RNA and protein changes following pRB- depletion and found that the pro-apoptotic genes were transcribed, but they were not translated into protein. A search for RNA-binding proteins (RBPs) that bound to and blocked the ribosome occupancy of these genes identified the Pumilio complex as direct translational regulators of RB-E2F pro-apoptotic mRNAs. Furthermore, co-deletion of the two core components of the Pumilio complex, PUM1 and PUM2, provoked the cell death of RB1-/- cancer cells via the translation of pro-apoptotic mRNAs. The human Pumilio complex is comprised of PUM and NANOS (NOS) proteins, however significant functional redundancy between the members of the PUM and NOS protein families has limited cellular and tumor studies of the Pumilio complex. To circumvent this issue, we have engineered human cells to express only a single PUM or NOS protein that we can specifically degrade utilizing a novel Auxin-inducible degron system. This technical advance has enabled us to conduct kinetic analysis of RNA stability, localization and translation initiation in the absence of PUM or NOS and to probe the role(s) of different Pumilio complexes in cellular regulation and tumorigenesis. In this new application we propose to: 1) Determine the mechanism(s) of Pumilio complex translational suppression of RB-E2F target pro-apoptotic genes, 2) Define the mechanism of Pumilio complex regulation in RB1 mutant Small Cell Lung Cancer (SCLC) cells, and 3) Determine the role of the Pumilio complex in the Rb1-/-; Trp53-/- mouse SCLC tumorigenesis model. These studies will identify the mechanism(s) that prevent the production of cell death components in highly aggressive and metastatic SCLC.
Loss of the RB1 tumor suppressor results in the transcription of cell death genes, but not a corresponding increase in cell death proteins. We found that the translation of these cell death mRNAs is silenced by the Pumilio complex of RNA binding protein(s). We propose to utilize a unique Auxin-inducible degron-mediated protein degradation system to dissect the detailed mechanism(s) associated with Pumilio complex regulation of pRB-regulated cell death transcripts in model human cells, Small Cell Lung Cancer (SCLC) cells and a well- defined murine SCLC model.
