Abstract 4852: Karyopherin-mediated regulation of autophagy in breast cancer cells
Authors: Thangavel Muthusamy, Mette P. Ibba
Journal: Cancer Research (2010)

Abstract Aim: Autophagy is a process of self-degradation of cellular components in which double-membrane autophagosomes sequester organelles or portions of cytosol and fuse with lysosomes for breakdown by resident hydrolases. Defective autophagy plays a significant role in human pathologies, including cancer. We present our current work that relates to the recent findings that protein synthesis and autophagy are reciprocally controlled by the same nutrient-sensitive signaling pathways. Our preliminary data suggest that human foreskin fibroblasts (HFF) and MCF7 cells with disrupted function of nucleocytoplasmic trafficking of transfer-tRNA (tRNA) show elevated levels of autophagy. The study aims to support the hypothesis that transfer-tRNA (tRNA) accumulation in the nucleus triggers an intracellular starvation response, which leads to autophagy upregulation. The work examines autophagy in the three breast cancer cells MCF7, MDA-MB-231 and SK-BR-3 knocked down for the tRNA-transporting karyopherin XPO-T and intends to decipher the molecular mechanism(s) behind the potential tRNA-mediated autophagic response and to study whether cell proliferation or cell death is affected. Methods: Knockdown of karyopherin XPO-T expression in MCF7, MDA-MB-231, SK-BR-3 is done by siRNA-mediated nucleofection followed by validation of reduced mRNA levels by western blot analysis and real-time PCR. Autophagy induction and progression are assessed by 1) western blot analysis and confocal microscopy of autophagic marker LC3, 2) flow cytometry to quantify GFP- tagged LC3 intensities and 3) transmission electron microscopy (TEM) to visualize autophagosomes. Activity of the nutrient-sensing GCN2 and mTOR pathways will be assessed by western blot analysis using antibodies towards P70S6k and eIF2α. Cell proliferation and cell death will be determined using methodologies such as cell proliferation kit MTT to monitor cell viability via ATP generation and trypan blue staining. Results and Conclusion: Our data suggest that karyopherin XPO-T, which transports newly synthesized mature tRNA from the nucleus to the cytosol, exerts a significant effect on autophagy in MCF7 and HFF cells. Autophagy upregulation due to reduced expression of XPO-T is suggested by changes in the levels of the lipidated form of LC3 (LC3-II) and by increased numbers of autophagosome-like structures in the cytosol. Interestingly, XPO-T knocked down in HFF cells show decreased mTOR Ser2448 phosphorylation and rpS6 Ser 235/236 phosphorylation, suggesting reduced activity of the mTOR pathway. Upregulation of autophagy supports cell survival. However, cell viability and proliferation is reduced approximately 30% in XPO-T dysfunctional HFF. In addition, GFP-LC3 intensities seem reduced 20% in XPO-T and TNPO-3 knocked down MCF7, which indicated faster lysosomal turnover of LC3 and elevated levels of autophagy progression in breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4852.