Abstract B72: Signaling and mRNA translation regulation in RSK-deficient LN-18 glioblastoma cell line

Abstract

Abstract The deregulation of p90 ribosomal S6 kinases (RSKs) seems to be responsible for different oncogenic outcomes in several types of tumors. The effects on mRNA translation regulation via RSKs in glioblastomas (GBMs) were not described so far; thus it is essential to understand the molecular pathways regulated by RSKs that control protein synthesis in GBM. Recent data from our group (unpublished) indicate that different GBM cell lines express distinct levels of the RSK1 isoform, while RSK2 levels are much less variable, and RSK3 and RSK4 are not detected. LN-18 cells show higher levels of RSK1 when compared to A172 or U-118 MG; thus CRISPR/Cas9 system technology was used in this cell line to generate RSK-deficient cells. Briefly, two different sets of small guide RNAs directed to both RSK1 and RSK2 were constructed. LN-18 cells were transfected to express Cas9 along with either one of the constructs targeting RSK1 and/or RSK2. Selection of individual cell clones was followed by Western blotting detection of RSK1 and RSK2 isoforms to identify RSK-deficient cells. We were able to efficiently generate clones with no detection of RSK1 (RSK1-/-), RSK2 (RSK2-/-) with both sets of sgRNAs and cells that had both isoforms knocked out (RSK1-/-/RSK2-/-) only with one of the sgRNA sets. Considering that mTORC1 signaling pathway is the main mediator of RSKs-dependent translation activation, the crosstalk between these pathways and its effects on translation levels were studied in RSKs-deficient cells. Therefore, cells were serum starved for 48 h and stimulated with phorbol-12-myristate-13-acetate (PMA) to activate the Ras-ERK1/2-RSK pathway. Signaling pathways involved in RSK and mTORC1 translation activation were assessed. Interestingly, RSK-deficient cells show lower levels of phosphorylation of Tuberous Sclerosis Complex 2 (TSC-2) at 1798 serine residue, confirming the importance of RSK phosphorylation on this residue and its relevance for evaluation of active RSK levels on cells. In order to describe the influence of RSK1 in the translation initiation rate of GBM cells, polysome profiling was assayed in LN-18, A172, and U-118 MG cells, containing high, low, or nondetected levels of RSK1, respectively. Treatment with PMA for 1, 2, or 4 hours affected translation in GBM cells in a different manner, with the cell lines that display lower levels of RSK1 being less sensitive to the treatment, when compared to LN-18 cells. Polysome profiling was also performed with CRISPR/Cas9-generated cells. RSK1-/- and RSK2-/- cells showed reduced translation initiation levels in basal conditions; however, translation initiation was stimulated after PMA treatment, probably because the remaining isoform is enough to elicit this response. Surprisingly, translation levels were comparable between control cells and RSK1-/-/RSK2-/- cells, suggesting the presence of a compensatory mechanism during the generation of double knockout cells. Of note, this same response could be observed in cells that display alterations in the elongation phase, as it can occur by the lack of phosphorylation of eEF2 kinase in the absence of RSKs. In conclusion, the generation of RSKs-deficient GBM-cell line by the CRISPR/Cas9 system is a useful model for studying and characterizing RSK effects on signaling and translation in GBM cells. Supported by FAPESP. Citation Format: Danielle Nascimento, Martín Roffé, Vilma Regina Martins. Signaling and mRNA translation regulation in RSK-deficient LN-18 glioblastoma cell line [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B72.