A CRISPR-edited isoform of the AMPK kinase LKB1 improves the response to cisplatin in A549 lung cancer cells

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Severino, Matheus Brandemarte ; Morelli, Ana Paula ; Pavan, Isadora Carolina Betim ; Mancini, Mariana Camargo Silva ; Gois, Mariana Marcela ; Borges, Rafael Junqueira ; Braga, Renata Rosseto ; da Silva, Luiz Guilherme Salvino ; Quintero-Ruiz, Nathalia ; Costa, Maira Maftoum ; Oliveira, Wesley de Lima ; Bezerra, Rosangela Maria Neves ; Ropelle, Eduardo Rochete ; Simabuco, Fernando Moreira

• Tipo de documento: Artigo Científico
• Fonte: Journal of Biological Chemistry; v. 301, n. 3, p. 21-pg., 2025-03-01.

Resumo

Lung cancer presents the highest mortality rate in the world when compared to other cancer types and often presents chemotherapy resistance to cisplatin. The A549 nonsmall cell lung cancer line is widely used as a model for lung adenocarcinoma studies since it presents a high proliferative rate and a nonsense mutation in the STK11 gene. The LKB1 protein, encoded by the STK11 gene, is one of the major regulators of cellular metabolism through AMPK activation under nutrient deprivation. Mutation in the STK11 gene in A549 cells potentiates cancer hallmarks, such as deregulation of cellular metabolism, aside from the Warburg effect, mTOR activation, autophagy inhibition, and NRF2 and redox activation. In this study, we investigated the integration of these pathways associated with the metabolism regulation by LKB1/AMPK to improve cisplatin response in the A549 cell line. We first used the CRISPR/Cas9 system to generate cell lines with a CRISPR-edited LKB1 isoform (called Super LKB1), achieved through the introduction of a +1 adenine insertion in the first exon of the STK11 gene after NHEJ-mediated repair. This insertion led to the expression of a higher molecular weight protein containing an alternative exon described in the Peutz-Jeghers Syndrome. Through metabolic regulation by Super LKB1 expression and AMPK activation, we found an increase in autophagy flux (LC3 GFP/RFP p < 0.05), as well as a reduction in the phosphorylation of mTORC1 downstream targets (S6K2 phospho-serine 423; p < 0.05; and S6 ribosomal protein phospho-serine 240/244; p < 0.03). The NRF2 protein exhibited increased levels and more nuclear localization in A549 WT cells compared to the edited cells (p < 0.01). We also observed lower levels of H2O2 in the WT A549 cells, as a possible result of NRF2 activation, and a higher requirement of cisplatin to achieve the IC50 (WT: 10 mu M; c2SL+: 5.5 mu M; c3SL+: 6 mu M). The data presented here suggests that the regulation of molecular pathways by the novel Super LKB1 in A549 cells related to metabolism, mTORC1, and autophagy promotes a better response of lung cancer cells to cisplatin. This NHEJ-CRISPR-based approach may be potentially used for lung cancer gene therapy. (AU)

• Processo FAPESP: 18/14818-9 - Estudo de alvos moleculares importantes para o controle do metabolismo em câncer: a via da mTOR/S6K com papel central
• Beneficiário: Fernando Moreira Simabuco
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores - Fase 2
• Processo FAPESP: 20/13660-2 - Edição do gene LKB1 por CRISPR/Cas9 em células de câncer de pulmão e avaliação de seu papel na resposta à metformina e cisplatina
• Beneficiário: Matheus Brandemarte Severino
• Modalidade de apoio: Bolsas no Brasil - Mestrado