Non-small Cell Lung Cancer (NSCLC) survival rates are dismal and βIII-tubulin is often aberrantly expressed in this disease1, where elevated expression is associated with chemotherapy resistance and tumour aggressiveness. Growing evidence links βIII-tubulin as a survival factor in cancer, where it may support survival of tumour cells in the harsh tumour microenvironment 2. This study aimed to investigate the role of βIII-tubulin in cellular survival and stress responses in NSCLC cells.
METHODS: βIII-tubulin shRNA or non-targeting control shRNA expressing NCI-H460 and A549 NSCLC cells were cultured in glucose-free media. Trypan Blue, BrdU and Annexin-V Apoptosis assays were used for proliferation and survival. Metabolic activity was measured using the XF24 Extracellular Flux Analyzer and radioactive glucose uptake assays. Akt signalling, ER stress response and autophagic activity were monitored by western blotting. XBP-1 splicing assays and immunofluorescence detected ER stress induction and autophagy, respectively.
RESULTS: βIII-tubulin influences glucose metabolism, with higher basal rates of glycolytic metabolism and glucose uptake in βIII-tubulin knockdown cells compared with control cells. Glucose depletion decreased cell number and viability in the βIII-tubulin knockdown cells and this was supported by increased cell death and decreased cell proliferation. Akt activation was dramatically reduced in cells with suppressed βIII-tubulin expression compared with controls in basal and glucose-starved conditions. Suppression of βIII-tubulin levels caused a greater induction of the ER stress response, as measured by levels of ER stress response proteins GRP78, ATF4 and CHOP and XBP-1 splicing assays. βIII-Tubulin co-immunoprecipitated with GRP78 under basal and glucose-starved conditions. Furthermore, βIII-tubulin knockdown altered autophagic flux in glucose-starved cells. These results indicate that βIII-tubulin modulates multiple stress response signalling pathways and promotes cell survival and proliferation in glucose starvation.
CONCLUSION: This study provides new mechanistic insight into how aberrant βIII-tubulin expression influences cell survival and poor prognosis in NSCLC.