Modulation of important non-coding RNAs and proteins in cancer with role in transcription and translation initiation regulation
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Date
2024-06-10
Authors
Κυριακόπουλος, Γεώργιος
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Abstract
Cancer progression involves the deregulation of several oncogenes and tumor suppressor genes, many of which participate directly or indirectly in signaling events that ultimately promote proliferation and survival. Two of the most commonly affected signaling pathways are the MAPK cascade and the PI3K/AKT/mTOR pathway. Both affect more than 200 downstream effectors and modulate almost every process of the cell, including transcription and translation. Transcription of coding RNAs by RNA pol II and non-coding RNAs by all three RNA polymerases leads, among others, to rewiring of protein synthesis. Eukaryotic translation has been considered one of the most energy consuming processes of the cells and, thus, it is tightly regulated. In order for a cell to become tumorigenic, translation reprogramming is necessary and is facilitated by complex regulatory networks that are mainly regulated by signaling pathways. Two of the most common oncogenes mutated in cancer are KRAS and BRAF, both of which regulate the MAPK cascade and the PI3K/AKT/mTOR signaling pathways. KRAS mutations are prevalent across numerous tumor types, including lung adenocarcinoma, which stands as one of the most frequently diagnosed cancers, often associated with unfavorable prognostic outcomes and limited efficacy of therapeutic interventions. Mutations in the BRAF gene primarily occur in melanoma but are also present in various other cancer types. Despite the availability of targeted treatments, melanoma cells rapidly develop resistance to BRAF inhibitors. These mutations can influence both transcription and translation processes, underscoring the need for a deeper understanding of their deregulation. To decipher the effects of KRASG12C on transcription and translation initiation in lung adenocarcinoma, generation of TetON-inducible KRASG12C stable A549 and CL1-5 cell lines using TALENs was performed. Further examination of the two cell lines revealed that in CL1-5 cells, cap-dependent translation was disrupted through the possible involvement of mTORC2 and NF-κB pathways. On the other hand, in A549 cells, cap-dependent translation was promoted through the activation of mTORC1, c-MYC, and the upregulation of the eIF4F complex. A decrease in the levels of eIF1, eIF5, and eIF5B in A549 cells indicated reduced fidelity in the initiation of protein synthesis. Puromycin staining and polysome profiling confirmed the increased translation activity in A549 cells compared to the compromised cap-dependent translation in CL1-5 cells. Notably, CL1-5 cells were able to recover their translation efficiency after extended activation of KRASG12C, but this recovery occurred via a route that did not involve mTORC1 or p70S6K. The expression profiles of specific tRNAs and tRFs also indicated variations in global protein synthesis rates. Finally, several mitochondrial tRFs were found modulated and correlated with deregulation of mitochondrial functions. In conclusion, in the first part of the present thesis, a divergent response is indicated among two epithelial-derived NSCLC cell lines upon induction of the same KRAS mutant, which affects the rewiring of protein synthesis, tRNA fragmentation and possibly mitochondrial integrity. At the second part of the present thesis, A375 and SKMEL5 melanoma cell lines resistant to vemurafenib (referred to as VR cell lines) were generated, and their IC50 values were determined. The development of resistance to vemurafenib resulted in a significant 10-fold increase in the IC50 of the A375VR and SKMEL5VR cell lines. These resistant cell lines exhibited continual activation of the MAPK pathway, even when exposed to high concentrations of vemurafenib. The mTOR kinase and its downstream substrates, S6K and 4EBPs, were differentially affected in the two VR cell lines, indicating differential effects on the signaling towards translation. The 60S/40S ratio indicated impaired ribosome biogenesis, which was correlated with the differential expression levels of the translation factor eIF6, suggesting its possible involvement in the acquired resistance to vemurafenib. To further investigate this, five melanoma cell lines (A375, SKMEL5, M1, M2 and M3) were edited to stably overexpress eIF6. These cells were analysed for changes in cell viability, proliferation rates, and migration, showing differential effects, possibly due to the different genomic context of each cell line. The response of the eFI6-overexpressing cells to vemurafenib was affected, with SKMEL5, M1 and M3 becoming more sensitive to BRAF inhibition, M2 cells more resistant, while A375 cells’ response remained unaffected. Higher 60S/40S ratios were observed in all cases, while key signaling effectors were differentially affected. The resistant eIF6-overexpressing M2 cells were correlated with lower phosphorylation levels of ERK1/2 kinase and higher total mTOR levels, while the opposite effects were observed in the sensitive eIF6-overexpressing SKMEL5, M1 and M3 cells. Taken together, the current thesis attempted to provide a comprehensive picture of the translation deregulation events governed by KRAS and BRAF mutants in lung adenocarcinoma and melanoma, respectively. The results show that KRASG12C can either promote or impair cap-dependent translation in two different lung adenocarcinoma cell lines. Finally, the present thesis further strengthens the significance of translation deregulation due to acquired resistance to vemurafenib in melanoma.
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Keywords
Cancer, Translation, Signaling pathways, KRAS mutation, Vemurafenib resistance