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E3 ubiquitin ligase FBXW7 enhances radiosensitivity of non-small cell lung cancer cells by inhibiting SOX9 regulation of CDKN1A through ubiquitination

Abstract

Non-small cell lung cancer (NSCLC) has high rates of morbidity and mortality. E3 ubiquitin ligase usually has antitumor effects. This study evaluated the mechanism of E3 ligase FBXW7 (F-box and WD repeat domain containing 7) in the radiosensitivity of NSCLC. NCI-H1299 and NCI-H1299R cells were irradiated by 0, 2, 4, and 6 Gy doses of X-ray, respectively. In addition to the measurement of cell proliferation, apoptosis, and γ-H2AX, FBXW7 expression was measured and the interaction between FBXW7 and SOX9 (SRY-box transcription factor 9) was evaluated. Ubiquitination level and protein stability of SOX9 were examined after FBXW7 overexpression. The binding relationship between SOX9 and CDKN1A (cyclin-dependent kinase inhibitor 1A) was verified. Xenograft tumor model was established to evaluate the effect of FBXW7 on radiosensitivity in vivo. FBXW7 was under-expressed in radioresistant cells. Overexpression of FBXW7 repressed NCI-H1299 and NCI-H1299R cell proliferation and colony formation and increased γ-H2AX-positive foci. Overexpression of FBXW7 increased the ubiquitination level and reduced the protein stability of SOX9. SOX9 bound to the CDKN1A promoter to inhibit CDKN1A expression. FBXW7 inhibited tumorigenesis and apoptosis and enhanced radiosensitivity of NSCLC cells in vivo via the SOX9/CDKN1A axis. Overall, FBXW7 inhibited SOX9 expression by promoting SOX9 ubiquitination and proteasome degradation, suppressing the binding of SOX9 to CDKN1A, and upregulating CDKN1A, thereby improving the radiosensitivity of NSCLC cells.

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Fig. 1: Resistant cells (NCI-H1299R) are more resistant to radiation than parental cells (NCI-H1299).
Fig. 2: Overexpression of FBXW7 enhanced the radiation sensitivity of NSCLC cells.
Fig. 3: FBXW7 interacted with SOX9 and promoted ubiquitination and proteasome degradation of SOX9.
Fig. 4: FBXW7 enhanced the radiosensitivity of NSCLC cells by inhibiting SOX9 expression.
Fig. 5: FBXW7 promoted CDKN1A expression via SOX9.
Fig. 6: FBXW7 enhanced the radiosensitivity of NSCLC cells by increasing CDKN1A expression.
Fig. 7: FBXW7 enhanced radiosensitivity of NSCLC cells in vivo via SOX9/CDKN1A.
Fig. 8: Molecular mechanism diagram.

Data availability

The data that support this study are available from the corresponding author upon reasonable request.

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Funding

The study was sponsored by Urumqi Science and Technology Bureau for the research on Changes of plasma EGFR gene mutation after TKI in advanced non-small cell lung cancer [Y161310003]. The funding organisations had no role in the concept, design, analysis or writing of the manuscript.

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HZ and XW: conceived and designed the experiments, and participated in design and coordination, and helped to draft the manuscript and revise the manuscript. XZ and SL: performed investigation, statistical analysis, and drafted the manuscript. GG and CL: contributed to the acquisition and analysis of data. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Chunling Liu.

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The authors declare no competing interests.

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This study was approved by the Ethical Committee of The Affiliated Tumor Hospital of Xinjiang Medical University (20200288). Animal experiments were conducted under the guidelines of the National Institutes of Health.

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Zhu, H., Wang, X., Zhou, X. et al. E3 ubiquitin ligase FBXW7 enhances radiosensitivity of non-small cell lung cancer cells by inhibiting SOX9 regulation of CDKN1A through ubiquitination. Lab Invest 102, 1203–1213 (2022). https://doi.org/10.1038/s41374-022-00812-9

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