Inhibition of protein translation under matrix-deprivation stress in breast cancer cells

Matrix-deprivation stress leads to cell-death by anoikis, whereas overcoming anoikis is critical for cancer metastasis. Work from our lab and others has identified a crucial role for the cellular energy sensor AMPK in anoikis-resistance, highlighting a key role for metabolic reprogramming in stress survival. Protein synthesis is a major energy-consuming process that is tightly regulated under stress. Although an increase in protein synthesis in AMPK-depleted experimentally-transformed MEFs has been associated with anoikis, the status and regulation of protein translation in epithelial-origin cancer cells facing matrix-detachment remains largely unknown. Our study shows that protein translation is mechanistically abrogated at both initiation and elongation stages by the activation of the unfolded protein response (UPR) pathway and inactivation of elongation factor eEF2, respectively. Additionally, we show inhibition of the mTORC1 pathway known for regulation of canonical protein synthesis. We further functionally assay this inhibition using SUnSET assay, which demonstrates repression of global protein synthesis in MDA-MB-231 and MCF7 breast cancer cells when subjected to matrix-deprivation. In order to gauge the translational status of matrix-deprived cancer cells, we undertook polysome profiling. Our data revealed reduced but continuous mRNA translation under matrix-deprivation stress. An integrated analysis of transcriptomic and proteomic data further identifies novel targets that may aid cellular adaptations to matrix-deprivation stress and can be explored for therapeutic intervention. To investigate global gene expression changes that occur following matrix-deprivation in MDA-MB-231 breast cancer cell line

基质缺失应激（matrix-deprivation stress）可通过失巢凋亡（anoikis）引发细胞死亡，而抑制失巢凋亡对于肿瘤转移至关重要。本实验室及其他团队的研究已证实，细胞能量感受器腺苷酸活化蛋白激酶（AMPK）在失巢凋亡抵抗中发挥关键作用，凸显了代谢重编程在应激存活过程中的核心地位。蛋白质合成是一类主要的耗能过程，在应激状态下受到严密调控。尽管在AMPK缺失的实验转化型小鼠胚胎成纤维细胞（MEFs）中，蛋白质合成的上调与失巢凋亡相关，但上皮来源肿瘤细胞在基质脱离状态下的蛋白质翻译状态及其调控机制仍尚未被充分阐明。本研究结果显示，蛋白质翻译在起始与延伸两个阶段分别通过未折叠蛋白反应（UPR）通路的激活，以及延伸因子eEF2的失活，被机制性阻断。此外，本研究还证实哺乳动物雷帕霉素靶蛋白复合物1（mTORC1）通路活性被抑制，而该通路正是经典蛋白质合成的调控通路。本研究进一步采用嘌呤霉素掺入检测法（SUnSET assay）对该抑制效应进行功能验证，结果显示，在MDA-MB-231与MCF7乳腺癌细胞中，基质缺失应激可导致整体蛋白质合成受到抑制。为了评估基质缺失状态下肿瘤细胞的翻译状态，本研究开展了多聚核糖体谱分析（polysome profiling）。本研究的数据显示，在基质缺失应激条件下，mRNA翻译水平有所降低但仍持续进行。对转录组与蛋白质组数据的整合分析进一步筛选出了潜在的新型靶点，这些靶点或可帮助肿瘤细胞适应基质缺失应激，有望为治疗干预提供新方向。为了探究MDA-MB-231乳腺癌细胞系在基质缺失后发生的整体基因表达变化。