Abstract 326: βIII-Tubulin and cell survival: Novel role in endoplasmic reticulum stress and autophagy in non-small cell lung cancer
Authors: Parker Amelia , McCarroll Joshua A. , Kavallaris Maria 
Published: May 2015

Non-small Cell Lung Cancer (NSCLC) survival rates are dismal and chemotherapy resistance is a significant barrier to effective treatment.

βIII-Tubulin is aberrantly expressed in a range of cancers, including NSCLC1. Elevated βIII-tubulin expression is associated with chemotherapy resistance and tumor aggressiveness in this disease. Growing evidence links βIII-tubulin as a survival factor in cancer, where it may support tumor cells in the harsh tumor microenvironment by regulating cellular stress responses2. This study aimed to investigate the role of βIII-tubulin in cellular survival and stress responses to glucose starvation in NSCLC cells.

METHODS: Previously described2 NCI-H460 cells with stable expression of βIII-tubulin shRNA (βIIIsh4 and βIIIsh59) or a non-targeting control shRNA (Ctrlsh1) were cultured in glucose-free media. Cell viability and proliferation were monitored over 14 days using Trypan Blue Dye Exclusion and Annexin-V Apoptosis assays. Metabolic activity was measured using the XF24 Extracellular Flux Analyzer. GRP78 protein expression was determined by western blotting. XBP-1 splicing assays were used to detect Endoplasmic Reticulum (ER) stress induction. Autophagic activity was examined using western blotting and immunofluorescence staining for LC3B.

RESULTS: Our results demonstrated that βIII-tubulin expression affects metabolic activity. Basal rates of glycolytic metabolism were higher in βIII-tubulin knockdown cells compared with control cells (p&lt;0.05). Glucose depletion led to decreased cell number and viability in the βIII-tubulin knockdown cells (p&lt;0.001) and this was supported by increased cell death in two independent βIII-tubulin shRNA clones (βIIIsh4 61.49±1.86%,

βIIIsh59 54.43±1.28% viability relative to Ctrlsh1 cells, p&lt;0.05). Moreover, βIII-tubulin knockdown markedly decreased GRP78 levels, a master regulator of the ER stress response and a pro-survival factor3. XBP-1 splicing assays confirmed the induction of ER stress upon glucose starvation, with elevated levels of XBP-1 mRNA in βIII-tubulin shRNA cells compared with controls. Furthermore, knockdown of βIII-tubulin altered autophagic flux under basal conditions, and this was further exacerbated by glucose starvation. These results strongly indicate that βIII-tubulin knockdown enhances induction of the ER stress response and autophagy in hypoglycemic conditions.

CONCLUSION: This study demonstrates for the first time that aberrant βIII-tubulin expression in NSCLC promotes cell survival in hypoglycaemic conditions and modulates the ER stress response and autophagy engaged during glucose starvation. These findings provide new mechanistic insight into the poor prognosis associated with high βIII-tubulin levels in NSCLC.

1 Kavallaris. Nature Rev Cancer, 10:194-204, 2010

2McCarroll et al., Cancer Res 70 :4995-5003, 2010

3Lee, Cancer Res, 67 :3496-3499, 2007

Citation Format: Amelia Parker, Joshua A. McCarroll, Maria Kavallaris. βIII-Tubulin and cell survival: Novel role in endoplasmic reticulum stress and autophagy in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 326. doi:10.1158/1538-7445.AM2014-326