The STK11 (LKB1) gene edition by CRISPR/Cas9 in non-small cells lung cancer A549, and the response to metformina and cisplatin

Author(s)

Matheus Brandemarte Severino

• Document type: Master's Dissertation
• Press: Limeira, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Aplicadas
• Defense date: 2023-12-15

Examining board members

Fernando Moreira Simabuco; Clarissa Ribeiro Reily Rocha; Augusto Ducati Luchessi

• Advisor: Fernando Moreira Simabuco

Abstract

INTRODUCTION: Cancer is a complex disease originating from the accumulation of mutations in cells. Among cancers, lung cancer has the highest mortality rates, with non-small cell lung cancer, particularly adenocarcinoma, being the most common subtype. A549 cells, bearing a significant mutation in the STK11 gene, often become resistant to cisplatin treatment, necessitating synergistic treatments. OBJECTIVES: The aim is to generate A549 cells with LKB1 expression and assess their response to cisplatin and the combination with metformin, comparing them to non-edited cells. METHODOLOGY: The CRISPR/Cas9 system was employed using a recombinant Cas9 and in vitro-synthesized guide RNA to edit the STK11 gene. Assays were performed to evaluate protein expression through Western Blotting and immunofluorescence, as well as analyses of glycolytic/aerobic metabolism and the redox system, including NRF2, antioxidant enzymes, and reactive species. RESULTS: Through non-homologous end joining (NHEJ), a new isoform of LKB1, referred to as "CRISPR-LKB1 (Super LKB1)" with a higher molecular weight, was generated. Cell lines expressing this isoform showed AMPK activation and regulations in mTORC, autophagy, and metabolism. Edited clones also exhibited better responses to cisplatin due to the accumulation of H2O2. CONCLUSION: The novel LKB1 isoform generated through the CRISPR/Cas9 system proved effective in activating the downstream target AMPK, leading to various cellular regulations and intensified apoptotic activity in the edited cells. Additionally, metformin enhanced cisplatin-induced apoptosis in non-edited cells, but it did not have additional effects on the edited cells (AU)

• FAPESP's process: 20/13660-2 - Genome editing of LKB1 gene by CRISPR/Cas9 in lung cancer cells and evaluation of its role in metformin and cisplatin response
• Grantee: Matheus Brandemarte Severino
• Support Opportunities: Scholarships in Brazil - Master