Investigation of the stress-induced translatome of matrix-deprived cancer cells

Abstract

Cancer remains one of the leading causes of death worldwide, in spite of all the ongoing research in the field of oncology. While there have been some breakthroughs in the past 50 years, metastatic tumors remain the bane of a patient’s life. The metastatic journey is replete with stresses that challenge the survival of these cancer cells. These range from hypoxia and nutrient deprivation at the primary site, shear stress in circulation to metastatic colonization at the secondary site. One of the primary stresses amongst many in this process, is the stress of matrix deprivation which is experienced by the cells as they invade and survive in circulation. While normal epithelial cells experience anoikis upon deprivation of matrix, cancer cells are anoikis-resistant. Our lab has previously uncovered important roles for AMPK, the cellular stress sensor kinase, in mediating anoikis resistance. As a stress sensor, AMPK inhibits anabolic pathways and promotes catabolic processes that generate energy to counter the bioenergetic stress. One of the major pathways inhibited by AMPK under stress is protein synthesis. While this inhibition of protein synthesis is characteristic of multiple other stresses like hypoxia, nutrient deprivation, oxidative stress and hyperosmotic stress, it remains relatively poorly explored in the stress of matrix deprivation. In this study, by exploration of the canonical stress response pathways impacting protein synthesis like UPR, mTORC1 pathway, AMPK-eEF2 axis and by functional assessment of protein translation, we illustrate that matrix deprivation is a bona-fide stress experienced by cancer cells. Upon investigation into the role of AMPK in this process, we show that while AMPK may important for the control of protein synthesis upon stress, there may also exist an AMPK independent translational response in matrix-deprived cancer cells. It has also been shown in the stress conditions like hypoxia or nutrient deprivation, that while protein synthesis is repressed, there is persistence of selective mRNA translation for adaptation and survival. However, such a detailed study on the existence of altered translatome has not been performed under matrix deprivation stress. Thus, we provide one of the first insights, into the possibility of selective translation under matrix deprivation stress by an integrated multi-omics analysis of transcriptomic and proteomic data on MDA-MB-231 invasive breast cancer cells cultured in adherent (attached) and suspension (detached) conditions. By this method, we identify potential targets and select a novel candidate protein CNBP, whose roles in breast cancer or matrix deprivation stress have not been elucidated yet. Herein, we also investigate into some possible roles of CNBP under matrix deprivation in breast cancer cells such as proliferation, stemness, inflammation and translation. Cumulatively, we provide conclusive evidence to show that epithelial-origin breast cancer cells (both established cell lines and patient-derived cancer cells) activate well-known stress-response pathways and consequently suppress protein synthesis upon encountering matrix-deprivation. We propose a relatively new hypotheses of its regulation under matrix detachment. We also delve into the identification of the altered translatome of these cancer cells under suspension stress and characterize a novel target for its roles in matrix-deprived cells. Eventually, these targets could guide adaptations to matrix deprivation and thus, be explored for therapeutic intervention.