Abstract 5484: Structural domains of βIII-Tubulin regulate multiple stress responses and influence cell growth and survival in glucose-deprived non-small cell lung cancer

Abstract

Abstract BACKGROUND: Non-small Cell Lung Cancer (NSCLC) survival rates are dismal and elevated βIII-tubulin expression is associated with chemotherapy resistance and tumor aggressiveness1. βIII-tubulin modulates glucose metabolism and stress response signaling to support cancer cell survival and proliferation in glucose starvation conditions in the tumor microenvironment2. βIII-Tubulin shares sequence similarity with other β-tubulin isotypes, differing in the C-terminal tail region of the protein, however the function of βIII-tubulin structural domains remain uncharacterized. This study investigated the importance of the βIII-tubulin body and tail structural domains in conferring survival advantage to glucose-starved NSCLC cells. METHODS: H460 NSCLC cells were gene-edited at the endogenous βIII-tubulin gene using zinc finger nuclease technology to replace the endogenous βIII-tubulin protein with either the full-length or truncated βIII-tubulin protein, or replacement of the βIII-tubulin body or tail with the βI-tubulin sequence, each fused to a GFP tag. Clones expressing the modified βIII-tubulin proteins were cultured in glucose-free conditions for 48h to 10 days and cell growth and viability were measured by Trypan Blue Dye Exclusion and Annexin-V apoptosis assay. The effect of the βIII-tubulin modifications on the endoplasmic reticulum (ER) stress response, autophagy and Akt signaling were examined by western blotting. RESULTS: Characterization of the gene-edited clones revealed that loss of the βIII-tubulin C-terminal tail or substitution of the βIII-tubulin body or tail sequence with that of the βI-tubulin sequence did not alter the microtubule architecture, cellular proliferation rate or viability in normal growth conditions. However, loss of the βIII-tubulin C-terminal tail significantly reduced the viability of H460 cells by 37 ± 0.05% in acute glucose starvation. Substitution of the βIII-tubulin body with the βI-tubulin body significantly reduced cell number by 53 ± 0.01% during prolonged glucose starvation. Loss of the βIII-tubulin C-terminal tail increased Akt activation and LC3B-II expression in glucose-rich conditions and potentiated the increase in ER stress response markers GRP78, ATF4 and CHOP in response to glucose starvation. These results indicate that the βIII-tubulin body and tail differentially confer a survival advantage to glucose-starved H460 cells and modulate multiple stress response signaling pathways in an isotype-dependent and independent manner. CONCLUSION: This study provides insight into the importance of βIII-tubulin structural domains in conferring βIII-tubulin function in NSCLC, thereby advancing our understanding of the role of tubulin isotypes in cell biology and the mechanisms contributing to poor patient outcome in this disease. 1 Kavallaris, Nat Rev Cancer 2010 2Parker et al., Carcinogenesis 2016 Citation Format: Amelia L. Parker, Wee S. Teo, Joshua A. McCarroll, Maria Kavallaris. Structural domains of βIII-Tubulin regulate multiple stress responses and influence cell growth and survival in glucose-deprived non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5484. doi:10.1158/1538-7445.AM2017-5484