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

Abstract

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.

Experimentally-driven mathematical model to understand the effects of matrix deprivation in breast cancer metastasis

“…The above experimental data obtained by previous work in our laboratory was used to initiate the development of breast cancer mathematical model. Previously, we also showed that increased levels of pAMPK upon matrix deprivation further leads to a decrease in pAkt 17 and mTOR activity 19 . The calibrated model (Supplementary Fig.…”

“…In vitro experimental data, retrieved from previously published work of our laboratory, was used for model calibration and validation. The data included the effects of matrix deprivation on pAkt, pAMPK, p-mTOR, p-S6K1 and metabolism in MDA-MB-231 cells 17,19 . It also consisted the effects of AMPK down-modulation 17 as compared to wild-type condition, upon matrix deprivation.…”

“…Additionally, we dissected that one of the causes for AMPK activation upon matrix deprivation is due to the spike in cytosolic calcium levels which contributes towards rapid and sustained activation of AMPK via CaMKK 18 . Moreover, we recently showed the inhibition of mTOR (a key player in protein synthesis) pathway upon matrix-deprivation 19 . Similarly, there may be several other proteins and feedback loops which may contribute towards the adaptation of cells to matrix deprivation.…”