LAT4 drives Temozolomide induced radiotherapy resistance in glioblastoma by enhancing mTOR pathway activation

Background As the strong adaptive radio-chemoresistance, GBM represents the worst prognosis form of primary malignant tumor within the central nervous system. Previous researches indistinctly focused on the mechanisms of resistance to X-ray or chemo regimen in isolation, however, it is still unknown if there exists the synergistic or interact effect between the above two kinds of resistances. Methods We established TMZ-resistant GBM cell lines (TMZ-R) by chronically exposing U87MG cell lines to TMZ, and DMSO was used as placebo control. In vivo and in vitro experiments verified the synergistic resistance of TMZ-R cells to radiotherapy. Cell proliferation and clonogenesis assay were used to detect cell tolerance to chemo- or ratio-treatment, immunofluorescence and comet assay to detect cell damage, and in vivo imaging to measure tumor size. By transcriptomics and series validation tests, LAT4 was identified to be associated with such TMZ induced radiotherapy resistance. The relationship between LAT4 and mTOR pathway activity was also analyzed. Finally, the effect of BCH, LAT inhibitor, combined with radiotherapy on GBM prognosis was verified in vivo. Results We have first confirmed that TMZ not only induces resistance to chemotherapy in GBM cells but also enhances their resistance to radiotherapy,which is a surprising discovery during the establishment of TMZ-resistant U87MG GBM cell lines. Comprehensive transcriptomic analysis identified amino acid metabolism as a potential key factor in radiotherapy resistance. It has been validated that the upregulation of LAT4, a member of leucine metabolism, subsequent to chemotherapy modulates the mechanistic target of mTOR pathway and leads to radiotherapy resistance both in vitro and in vivo. Importantly, the application of inhibitors targeting leucine metabolism has been demonstrated to restore the sensitivity of these cells to radiotherapy, highlighting a potential therapeutic strategy for overcoming resistance in GBM. Conclusions Our study first discover the synergistic effect between tumor resistance to chemotherapy and radiotherapy. Our study highlights the critical role of LAT4 in activating the mTOR pathway and such resistance interaction. Targeting LAT4 and mTOR pathway will improve treatment sensitivity of GBM, especially in recurrent tumors.