TGM2-Mediated Autophagy Contributes to the Radio-Resistance of Non-Small Cell Lung Cancer Stem-like Cells

<b>Objectives</b>: Cancer cells with 'stemness' are generally resistant to chemoradiotherapy. This study aims to compare the differences in radiation sensitivity of A549 and CD44⁺A549 stem-like cells to X-rays and carbon ion radiation (C-ions), and to find a target that can kill cancer stem-like cells (CSCs) of non-small cell lung cancer (NSCLC). <b>Methods</b>: The study used two cell lines (A549 and CD44⁺A549). The tumorigenicity of cells was tested with animal experiments. The cells were irradiated with X-rays and C-ions. Cell viability was detected using the CCK-8 and EdU assay. A liquid chromatograph-mass spectrometer (LC-MS) helped detect metabolic differences. Protein and mRNA expression were detected using a Western blot, reverse transcription-quantitative (RT-qPCR), and PCR array. The autophagic activity was monitored with a CYTO-ID^® Autophagy Detection Kit 2.0. Immunofluorescence and co-immunoprecipitation helped to observe the localization and interaction relationships. <b>Results:</b> First, we verified the radio-resistance of CD44⁺A549 stem-like cells. LC-MS indicated the difference in autophagy between the two cells, followed by establishing a correlation between the radio-resistance and autophagy. Subsequently, the PCR array proved that TGM2 is significantly upregulated in CD44⁺A549 stem-like cells. Moreover, the TGM2 knockdown by small interfering RNA could decrease the radio-resistance of CD44⁺A549 cells. Bioinformatic analyses and experiments showed that TGM2 is correlated with the expression of CD44 and LC3B. Additionally, TGM2 could directly interact with LC3B. <b>Conclusions:</b> We established the CD44-TGM2-LC3 axis: CD44 mediates radio-resistance of CD44⁺A549 stem-like cells through TGM2 regulation of autophagy. Our study may provide new biomarkers and strategies to alleviate the radio-resistance of CSCs in NSCLC.