Day 0: Seeded human primary fibroblasts (HCA2 cell line).

1. Translation Regulation Machinery is Essential for the Senescence-Associated Secretory Phenotype Mauricio Borda, Rémi-Martin Laberge, and Judith Campisi Buck Institute for Age Research, 8001 Redwood Blvd., Novato, CA 94945 Abstract Aging is characterized by the degeneration of tissues in the body and by hyperplastic pathologies such as cancer. These seemingly opposite pathologies begin to occur more frequently around halfway through an organism’s lifespan and increase exponentially with every year. Cellular senescence, the permanent halting of proliferation due to potential oncogenic stresses, also increases with age and appears to link the two. Senescent cells prevent tumorigenesis by ceasing to divide. However, when becoming senescent, cells secrete pro-inflammatory cytokines, chemokines, growth factors, and proteases. This secretion is known as the senescence-associated secretory phenotype (SASP) and may have a role in the development of age related pathologies. The mammalian target of rapamycin (mTOR), a nutrient sensor, has been found to contribute to production of the SASP. The inhibition of mTOR with the lifespan enhancer rapamycin leads to a decrease in the SASP. In the present study, we investigated the importance of translation regulation machinery downstream of mTOR on the SASP. Using a shRNA approach, we found that downregulating the transcript levels of EIF4B, EIF4E, RPTOR, RPS6KA1, RPS6KA3, RPS6, RPS6KB1, and RPS6KB2 decreased the SASP. These results show that downregulating translational machinery leads to a decrease in the SASP. Better understanding of the mechanisms of this secretion may have clinical applications as it opens a new avenue for decreasing SASP by targeting, for instance, any of the translational machinery components. Senescent cells increase with age and in response to cancer treatments such as chemotherapy and radiotherapy. Significantly decreasing the level of the SASP produced by senescent cells may open up new ways to lessen the harmful effects associated with current cancer treatments and aging in general. Figure 3: Extracellular growth factors bind to a membrane receptor. This begins a protein cascade through PI3K and RAS pathways ultimately leading to the activation of mTORC1, RSK1, and RSK2. Nutrients and energy can also activate mTORC1. mTORC1 is a complex composed of various proteins including mTOR and Raptor. mTORC1 phosphorolates S6K1 and S6K2 which, in turn, phosphorolate eIF4B and RPS6 which are important for translation. RSK1 and RSK2 also activate eIF4B and RPS6. eIF4E is inhibited by 4EBP1 but when mTORC1 phosphorolates 4EBP1, eIF4E disassociates and becomes active. eIF4E is a major player in cap-dependent translation. Through some mechanism still not well understood, NF-κB gets activated which promotes the secretion of SASP. Results Methods The data reveal that there is an increase in the SASP when control cells treated with shGFP were irradiated and rendered senescent. After analyzing transcript levels, there is approximately a 38 fold increase in IL-6, 51 fold increase in IL-1α, and 122 fold increase in CXCL1 (Figure 1). Genes associated with translation did not have a large expression variation. Of these, RPTOR had the highest with less than a 3.5 fold increase (Figure 1). Downregulation of EIF4B, EIF4E, RPTOR, RPS6KA1, RPS6KA3, RPS6, RPS6KB1, and RPS6KB2 decreased the SASP to a large extent (Figure 2). Day 0: Seeded human primary fibroblasts (HCA2 cell line). Day 1: Infected cells using various lentiviruses encoding for short hairpins to knock down genes of interest. Day 3: Added puromycin to select for the infected cells. Day 7: The cells were split. Some had mRNA extracted, were reverse transcribed to cDNA and had qPCR run. The others were reseeded. Day 8: The seeded cells were x-ray irradiated rendering them senescent. Day 14: The senescent cells had qPCR performed as for nonsenescent cells. NonSen qPCR Infection Selection X-ray Sen qPCR 14 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Days Discussion In this study, we investigated the importance of the translational regulation machinery downstream of mTOR on the SASP. With the use shRNA inserted via lentiviruses, we found that downregulating the transcript levels of EIF4B, EIF4E, RPTOR, RPS6KA1, RPS6KA3, RPS6, RPS6KB1, and RPS6KB2 decreased the SASP. EIF4EBP1, when bound to eIF4E inhibits eIF4E from proceeding with translation (Figure 3). This would explain why we witnessed an ineffective decrease in the SASP compared to the other downregulated genes. by targeting genes involved in translational regulation. As expected, EIF4EBP1, a gene that reduces translation, did not decrease the SASP as effectively as the other genes when downregulated. In conclusion, the data suggest that sites other than mTORC1 can be targeted to decrease the level of the SASP. Specifically, genes involved in translation regulation appear to make promising candidates. Chart 1. Label in 24pt Calibri. Figure 2: HCA2 cells were infected with lentiviruses encoding short hairpin RNA in order to lower transcript levels of genes regulating translation. Cells were exposed to X-ray irradiation. qPCR was performed on senescent cells to measure the level of translational regulators compared to the level of the SASP. IL-6, IL-1 α, and CXCL1 are all markers for the SASP. The first data point in each group has senescent cells encoding shGFP, all other values were normalized to this control. The other points in the group show the mRNA levels of cells encoding for short hairpins of other genes. The numbers after the short hairpins represent different hairpins targeting the same gene, hence the varied level of knockdown. Figure 1: qPCR analysis of control senescent cells infected with shGFP. These values have been normalized by comparing to the same gene in nonsenescent cells infected with GFP. Genes associated with SASP had 6 duplicates. Error bars signify standard deviation.