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CRISPR/Cas-Mediated Knockdown of PD-L1 and KRAS in Lung Cancer Cells
Summer A Abounar1, Nefertiti A El-Nikhely1,2, Kati Turkowski3
1Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Alexandria 21526, Egypt.
Abstract:
Cancer cells can escape death and surveillance by the host immune system in various ways. Programmed cell death ligand 1 (PD-L1) is a transmembrane protein that is expressed by most cell types, including cancer cells, and can provide an inhibitory signal to its receptor PD-1, which is expressed on the surface of activated T cells, impairing the immune response. PD-L1/PD-1-mediated immune evasion is observed in several KRAS-mutated cancers. In the current study, we used the CRISPR/Cas9 system to knock down PD-L1 and KRAS in adenocarcinoma lung cells (A549 and H1975). Knockdown of PD-L1 was validated by qPCR and coculture with lymphocytes. The cells were functionally analyzed for cell cycle, migration and apoptosis. In addition, the effects of PD-L1 and KRAS downregulation on chemotherapy sensitivity and expression of inflammatory markers were investigated. Suppression of PD-L1 and KRAS led to a slowdown of the cell cycle in the G0/G1 phase and reduced migration, increased sensitivity to chemotherapy and triggered apoptosis of cancer cells. In addition, the conditioned medium of the modulated cells significantly affected the native cancer cells and reduced their viability and drug resistance. Our study suggests that dual silencing of PD-L1 and KRAS by CRISPR/Cas9 may be a promising therapeutic approach for the treatment of lung cancer.
Insights
Dual silencing of programmed cell death ligand 1 (PD-L1) and KRAS in lung cancer cells using CRISPR/Cas9 inhibits cell growth and enhances chemotherapy sensitivity. This approach shows promise for treating lung cancer by overcoming immune evasion.
Area of Science:
- Molecular biology
- Immunology
- Oncology
Background:
- Cancer cells evade immune surveillance through mechanisms like PD-L1/PD-1 interaction.
- This immune evasion is prevalent in KRAS-mutated cancers, hindering T cell activity.
- Targeting PD-L1 and KRAS is crucial for effective cancer therapy.
Purpose of the Study:
- To investigate the therapeutic potential of simultaneously downregulating PD-L1 and KRAS in lung cancer.
- To evaluate the effects of dual gene silencing on cancer cell behavior and treatment response.
Main Methods:
- Utilized CRISPR/Cas9 gene editing to knock down PD-L1 and KRAS in lung adenocarcinoma cell lines (A549 and H1975).
- Validated PD-L1 knockdown using qPCR and lymphocyte coculture assays.
- Assessed functional impacts on cell cycle, migration, apoptosis, and chemotherapy sensitivity.
Main Results:
- Suppression of PD-L1 and KRAS led to G0/G1 cell cycle arrest and reduced cell migration.
- Downregulation significantly increased sensitivity to chemotherapy and induced cancer cell apoptosis.
- Conditioned medium from silenced cells reduced viability and drug resistance in native cancer cells.
Conclusions:
- Dual silencing of PD-L1 and KRAS via CRISPR/Cas9 presents a potential therapeutic strategy for lung cancer.
- This approach effectively combats immune evasion and enhances treatment efficacy.
- Further research is warranted to explore this dual-targeting strategy in clinical settings.
