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Trna Biogenesis and Specific Aminoacyl-Trna Synthetases Regulate Senescence Stability Under the Control of Mtor

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Trna Biogenesis and Specific Aminoacyl-Trna Synthetases Regulate Senescence Stability Under the Control of Mtor bioRxiv preprint doi: https://doi.org/10.1101/2020.04.30.068114; this version posted May 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. tRNA biogenesis and specific Aminoacyl-tRNA Synthetases regulate senescence stability under the control of mTOR Jordan Guillon, Bertrand Toutain, Alice Boissard, Catherine Guette and Olivier Coqueret#. ICO Cancer Center, CRCINA, INSERM, Université de Nantes,Université d’Angers, France. # Corresponding author: Olivier Coqueret [email protected] ICO Cancer Center INSERM U1232, 15 rue Boquel, 49055 ANGERS, France Running title: tRNA deregulation during senescence Keywords: Chemoresistance senescence, mTOR, tRNA, ER stress. 1! bioRxiv preprint doi: https://doi.org/10.1101/2020.04.30.068114; this version posted May 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ABSTRACT nuclear envelope which allows leakage of chromatin fragments within the cytoplasm. These abnormal fragments are detected by the cGAS- Senescence normally prevents the STING DNA sensing pathway which then activates propagation of abnormal cells but is also NF-kB and induces the production of a specific associated with cancer progression and secretome known as the SASP (Senescence- chemotherapy resistance. These contradictory Associated Secretory Phenotype). Mainly effects are related to the stability of epigenetic composed of cytokines and chemokines, this marks and to the senescence-associated secretory secretome maintains senescence and attracts phenotype (SASP). The SASP reinforces the immune cells which then eliminate the senescent proliferative arrest but also induces tumor populations (4-7). Thus, through the up-regulation growth and inflammation during aging. of the p53 and Rb pathways and the activation of Senescence is therefore much more immune responses, senescence prevents the heterogeneous than initially thought. How this propagation of abnormal cells. response varies is not really understood. Although initially described as a definitive Using experimental models of proliferative arrest, we and others have recently senescence escape, we now described that the shown that some cells can escape senescence, deregulation of tRNA biogenesis affects the indicating that distinct stages of light and deep stability of this suppression, leading to senescence should be distinguished (2,7-9). This chemotherapy resistance. Proteomic analyses heterogeneity can be explained by a variable showed that several aminoacyl-tRNA synthetases expression of p16 which is necessary for were down-regulated in senescent cells. tRNA senescence maintenance. The stability of transcription was also inhibited as a consequence senescence also implies that the compaction of of a reduced DNA binding of the type III RNA proliferative genes is maintained within the polymerase. Reducing RNA Pol III activity by SAHFs. Recent results have reported that BRF1 depletion maintained senescence and H3K9Me3 repressive marks can be removed by the blocked cell persistence. Results showed that the JMJD2C and LSD1 demethylases and that this YARS1 and LARS1 aminoacyl-tRNA induces senescence escape (10). The HIRA histone synthetases were necessary for cell emergence chaperone also plays a key role in the deposition of and that their corresponding tRNA-Leu-CAA the histones H3.3 and H4 into the chromatin. Its and tRNA-Tyr-GTA were up-regulated in down-regulation allows senescence escape, persistent cells. On the contrary, the CARS1 indicating again that the stability of epigenetic ligase had no effect on persistence and the marks plays a key role in the maintenance of the expression of the corresponding tRNA-Cys was suppressive arrest. The dynamic nature of not modified. Results also showed that these senescence is also explained by the variability of tRNAs were regulated by mTOR and that this the SASP. Initially described as beneficial, several abnormal tRNA biogenesis induced an ER stress studies have reported that its composition varies which was resolved by the kinase during and that this secretome can also enhance chemotherapy escape. inflammation or tumor progression (11-13). The Overall, these findings highlight a new reason for this variability is not really understood. regulation of tRNA biology during senescence Cancer cell lines also represent an interesting and suggest that specific tRNAs and ligases model of an incomplete senescence response. We contribute to the strength and heterogeneity of and others have shown that this suppression this suppression and to chemotherapy resistance. functions as an adaptive mechanism in response to chemotherapy-induced -senescence (CIS) (7,9). We have described that cancer cells can escape senescence and emerge as more transformed cells INTRODUCTION that resist anoikis and are more invasive (14-17). In addition, we have also shown that cells having an Senescence induces a definitive incomplete senescence response are characterized proliferative arrest in response to telomere by a reduced expression of CD47 (17). As shortening, oncogenes or chemotherapy (1,2). This previously proposed (18,19), this indicates that tumor suppression is most of the time induced by senescent populations are heterogeneous and can be DNA damage and activation of the p53-p21 and identified by cell surface receptors. p16-Rb pathways. A definitive proliferative arrest Taken together, these studies indicate that is then maintained by the Rb-mediated compaction senescence is much more dynamic than initially of proliferative genes within heterochromatin foci thought and that a better characterization of these or SAHFs (Senescence Associated arrested cells is necessary. At the single cell level, Heterochromatin Foci) (3). Senescence is also any modification of epigenetic marks, of the SASP characterized by an increased permeability of the composition or any variability of the oncogenic 2! bioRxiv preprint doi: https://doi.org/10.1101/2020.04.30.068114; this version posted May 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. background will generate heterogeneous analysis indicated that Myc and E2Fs proliferative populations. In this study, we pursued our pathways were reactivated as expected. experiments with the aim of characterizing the Interestingly, a significant up-regulation of mTOR signaling pathways involved in the maintenance of signaling was also detected (Figure 1D). We senescence. We describe in this work that mTOR focused on this kinase since it plays a key role activates tRNAs synthesis during senescence during senescence (21-25). Western blot analysis escape. During the initial steps of this suppressive confirmed that mTOR was up-regulated in LS174T arrest, the transcription of the type III RNA and MCF7 cells following p21 inhibition, as shown polymerase is down-regulated and tRNA synthesis by the phosphorylation of the S6 ribosomal protein, is then reactivated during senescence escape. one of its main target (Figure 1E). We then asked if Depending on the experimental model, results mTOR was involved in CIS escape by treating the showed that specific tRNAs were reactivated, such cells at the beginning of emergence with torin-1 as the tRNALeu-CAA and tRNATyr-GTA tRNAs in and rapamycin, two common drugs used to emergent colorectal cells or breast organoids. In inactivate this kinase. These two inhibitors addition, specific aminoacyl-tRNA synthetases significantly blocked CIS escape following p21 (ARS) such as the Leucyl-and Tyrosyl-tRNA inactivation (Figure 1F). Emergent cells were more ligases were necessary for senescence escape. Our sensitive to mTOR inhibition than parental, results also indicate that this deregulation of tRNA untreated cells (Supplementary Figure 1B). In synthesis led to the activation of the unfolded addition, Rb phosphorylation or cyclin A protein response (UPR) and that this tRNA- expression were not inhibited by the two drugs mediated ER stress is resolved by mTOR to allow (Figure 1G). We then determined if mTOR was also senescence escape. involved in spontaneous CIS escape, in the absence Altogether, these results indicate that of p21 manipulation. Western blot analysis specific pools of tRNAs or ARSs regulate the indicated that the kinase was activated at the early outcome of this suppressive response and of steps of emergence, two days after serum release chemotherapy. We propose that different types of (Figure 1H). In this condition, torin-1 or rapamycin senescence, replicative, oncogenic or mediated by inhibited its activity and significantly blocked CIS chemotherapy might lead to the expression of escape, both in LS174T and MCF7 cells (Figure 1H different pools of tRNA and ARSs. This could and I). partly explain the variability of the SASP and the Altogether these observations indicate deleterious effects of senescence in response to mTOR is necessary for CIS escape, either during treatment. spontaneous emergence or following p21 inactivation. RESULTS Activating ER stress prevents CIS escape mTOR inhibition prevents CIS escape Interestingly, the mass spectrometry analysis also detected a significant deregulation of In breast and colorectal cell lines that do the Unfolded Protein Response (UPR) following not express p16INK4, we have previously
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