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Published on: July 12, 2022
SRSF3 knockdown-induced cellular senescence as a possible therapeutic strategy for non-small cell lung cancer
Shinji Nakamichi1, Natalia von Muhlinen1, Leo Yamada1
1Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Abstract:
Tyrosine kinase (TK) inhibitors improve clinical outcomes in non-small cell lung cancer (NSCLC) with targetable mutations. However, such NSCLC cases only consist of about 50% in the western populations. This study, for the first time in NSCLC cells including those without a targetable TK mutation, explores a tumor-suppressive activity of siRNA knockdown of a splicing factor SRSF3, which was reportedly effective in other cancer cell types. The knockdown of SRSF3 increased cellular senescence, indicated by senescence-associated β-galactosidase activity and reduced cell proliferation, in all NSCLC cell lines examined, including A549 (no TK mutation; TP53 wild-type), NCI-H1975 (EGFR L858R/T790M; TP53 R273H mutant), NCI-H322 (no TK mutation; TP53 R248L mutant) and NCI-H596 (no TK mutation; TP53 G245C mutant). An increase in apoptotic cleavage of caspase-3 and poly(ADP-ribose) polymerase was also observed in A549 cells. p53β, a tumor-suppressive p53 isoform generated via alternative mRNA splicing, was upregulated by SRSF3 knockdown, as previously reported in normal fibroblasts. However, neither cellular senescence nor apoptosis was increased by overexpression of p53β, suggesting no or minimum contribution of this p53 isoform to the tumor-suppressive activity of SRSF3 knockdown in NSCLC cells. Our gene expression assay indicated that the SRSF3 knockdown-induced senescence in NSCLC cells may be mediated by downregulation of TOP2A, UBE2C or ASPM, which are known to be oncogenic and are associated with poor patient prognosis. We also generated SRSF3 siRNA-encapsulating lipid nanoparticles as a future therapeutic tool. This study suggests a therapeutic strategy for NSCLC irrespective of the mutation status of TP53 and TK-encoding genes.
Insights
Silencing SRSF3 triggers tumor suppression in non-small cell lung cancer (NSCLC) cells, regardless of targetable mutations. This approach induces senescence and apoptosis, offering a potential new therapy for NSCLC patients.
Area of Science:
- Molecular Biology
- Oncology
- Cancer Genetics
Background:
- Tyrosine kinase (TK) inhibitors are effective for non-small cell lung cancer (NSCLC) with targetable mutations, but these mutations are present in only about 50% of Western patients.
- Alternative splicing factors, like SRSF3, play roles in various cancers, but their tumor-suppressive potential in NSCLC, particularly in cells lacking targetable TK mutations, remains largely unexplored.
Purpose of the Study:
- To investigate the tumor-suppressive activity of SRSF3 knockdown in diverse NSCLC cell lines, including those without targetable TK mutations.
- To elucidate the mechanisms underlying SRSF3 knockdown-induced tumor suppression, including cellular senescence, apoptosis, and the role of p53 isoforms and specific gene pathways.
Main Methods:
- Utilized siRNA to knockdown SRSF3 in various NSCLC cell lines (A549, NCI-H1975, NCI-H322, NCI-H596).
- Assessed cellular senescence via senescence-associated β-galactosidase activity and measured cell proliferation.
- Quantified apoptosis by detecting cleaved caspase-3 and poly(ADP-ribose) polymerase.
- Analyzed the expression of p53 isoforms and oncogenic genes (TOP2A, UBE2C, ASPM) using gene expression assays.
- Developed SRSF3 siRNA-encapsulating lipid nanoparticles as a potential therapeutic delivery system.
Main Results:
- SRSF3 knockdown induced cellular senescence and reduced proliferation in all tested NSCLC cell lines, irrespective of TK mutation or TP53 status.
- Apoptosis was observed in A549 cells upon SRSF3 knockdown.
- While SRSF3 knockdown upregulated the tumor-suppressive p53 isoform p53β, its overexpression did not induce senescence or apoptosis, indicating a minimal role.
- Gene expression analysis suggested that SRSF3 knockdown-induced senescence may involve the downregulation of oncogenic genes TOP2A, UBE2C, or ASPM.
- Functional SRSF3 siRNA-encapsulating lipid nanoparticles were successfully generated.
Conclusions:
- SRSF3 knockdown exhibits significant tumor-suppressive activity in NSCLC cells, offering a potential therapeutic strategy applicable to a broad range of patients, including those lacking targetable mutations.
- The observed effects are primarily mediated by inducing cellular senescence, potentially through the downregulation of key oncogenic genes, rather than through the p53β isoform.
- The development of siRNA-encapsulating nanoparticles presents a promising avenue for future NSCLC therapy development.
