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Updated: Jan 11, 2026
Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells
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, United States.
Abstract:
Tyrosine kinase (TK) inhibitors improve clinical outcomes in non-small cell lung cancer (NSCLC) with targetable mutations. However, such NSCLC cases account for only about 50% in the western populations. Inhibition of the splicing factor SRSF3 has been reported to be tumor-suppressive in other cancer cell types. This study for the first time explores the tumor-suppressive activity of siRNA knockdown of SRSF3 in NSCLC cells. The cell lines used were 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). In all these cell lines, SRSF3 knockdown increased cellular senescence, as indicated by increased senescence-associated β-galactosidase activity and reduced cell proliferation. In A549 cells, increased apoptotic cleavage of caspase-3 and poly(ADP-ribose) polymerase was also observed. A tumor-suppressive p53 isoform, p53β, was shown to be upregulated by SRSF3 knockdown. However, overexpression of p53β did not induce cellular senescence or apoptosis, suggesting that this p53 isoform is not a primary effector of SRSF3 knockdown in NSCLC cells. Gene expression analyses suggested that the SRSF3 knockdown-induced senescence in NSCLC cells may be mediated by the downregulation of TOP2A, UBE2C, or ASPM, which are known oncogenic factors associated with poor patient prognosis. We also generated SRSF3 siRNA-encapsulating lipid nanoparticles as a future therapeutic tool. This study proposes a therapeutic strategy for NSCLC that is independent of the mutation status of TP53 and TK-encoding genes.
Insights
Targeting SRSF3 with siRNA shows tumor-suppressive effects in non-small cell lung cancer (NSCLC) by inducing senescence. This novel approach offers a potential therapeutic strategy for NSCLC, regardless of mutation status.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Genetics
Background:
- Tyrosine kinase (TK) inhibitors benefit a subset of non-small cell lung cancer (NSCLC) patients with targetable mutations.
- A significant portion of NSCLC cases lack these actionable mutations, necessitating alternative therapeutic strategies.
- The splicing factor SRSF3 has demonstrated tumor-suppressive roles in various cancer types.
Purpose of the Study:
- To investigate the tumor-suppressive potential of SRSF3 knockdown in diverse non-small cell lung cancer (NSCLC) cell lines.
- To elucidate the molecular mechanisms underlying SRSF3 knockdown-induced anti-cancer effects in NSCLC.
- To develop a novel, mutation-independent therapeutic strategy for NSCLC.
Main Methods:
- Utilized siRNA to knockdown SRSF3 expression in multiple NSCLC cell lines (A549, NCI-H1975, NCI-H322, NCI-H596).
- Assessed cellular senescence via senescence-associated β-galactosidase activity and cell proliferation assays.
- Analyzed apoptosis markers (caspase-3, PARP), p53 isoform expression, and gene expression profiles of oncogenic factors (TOP2A, UBE2C, ASPM).
- Developed SRSF3 siRNA-encapsulating lipid nanoparticles for potential therapeutic delivery.
Main Results:
- SRSF3 knockdown consistently induced cellular senescence and reduced proliferation across all tested NSCLC cell lines.
- Apoptosis was significantly increased in A549 cells following SRSF3 knockdown.
- SRSF3 knockdown upregulated the tumor-suppressive p53 isoform, p53β, though p53β overexpression alone did not induce senescence or apoptosis.
- Gene expression analysis indicated that SRSF3 knockdown-induced senescence may involve the downregulation of oncogenes TOP2A, UBE2C, or ASPM.
- Functional SRSF3 siRNA-loaded lipid nanoparticles were successfully generated.
Conclusions:
- SRSF3 functions as an oncogene in NSCLC, and its inhibition triggers tumor-suppressive mechanisms including senescence.
- The observed effects are independent of TP53 and tyrosine kinase mutation status, offering a broadly applicable therapeutic avenue.
- Downregulation of specific oncogenes like TOP2A, UBE2C, or ASPM may mediate the senescence induced by SRSF3 inhibition.
- SRSF3 siRNA delivered via lipid nanoparticles represents a promising future therapeutic strategy for NSCLC.
