Temporal Quantitative Proteomics Reveals Proteomic and Phosphoproteomic Alterations Associated with Adaptive Response to Hypoxia in Melanoma Cells

Abstract

Hypoxia is a common feature in various solid tumours, including melanoma. Cancer cells in hypoxic environments are resistant to both chemotherapy and radiation. Hypoxia is also associated with immune suppression. Identification of proteins and pathways that regulate cancer cell survival in hypoxic environments can reveal potential vulnerabilities that can be exploited to improve the efficacy of anticancer therapies. We carried out temporal proteomic and phosphoproteomic profiling in melanoma cell lines to identify hypoxia-induced protein expression and phosphorylation changes. By employing a TMT-based quantitative proteomics strategy, we report the identification and quantitation of >7000 proteins and >10,000 phosphosites in melanoma cell lines grown in hypoxia. Proteomics data show metabolic reprogramming as one of the prominent adaptive responses in hypoxia. We identify several novel hypoxia-mediated phosphorylation changes that have not been reported before. They reveal kinase signalling pathways that are potentially involved in modulating cellular response to hypoxia. In addition to known protein expression changes, we identify several novel proteomic alterations associated with adaptive response to hypoxia. We show that cancer cells require the ubiquitin–proteasome system to survive in both normoxia and hypoxia. Inhibition of proteasome activity affects cell survival and may provide a novel therapeutic avenue to target cancer cells in hypoxia. Our study can serve as a valuable resource to pursue novel candidates to target hypoxia in cancers and improve the efficacy of anticancer therapies.

Phosphoproteomic landscape of pseudorabies virus infection reveals multiple potential antiviral targets

“…After sonication, the samples were centrifuged at 12,000 rpm for 20 minutes to remove insoluble fractions. Protein concentration was determined using a BCA Assay Kit (Thermo Fisher Scientific, USA) as described before ( 35 ). Then, 10 µg of protein from each sample was collected and separated using SDS-PAGE.…”

Systems approaches to understand oxygen sensing: how multi-omics has driven advances in understanding oxygen-based signalling

“…Phosphorylation is a crucial PTM regulating a many of the intra- and intercellular cell signalling pathways induced in normal and malignant hypoxic cells. As discussed, changes in phosphorylation states play key roles in regulating HIF signalling; however, large-scale changes in the phosphoproteome are observed in cells exposed to hypoxic stress [ 107 , 108 ]. A recent phosphoproteomic study analysing the hypoxia-induced phosphoproteome from four distinct melanoma cell lines revealed both a core set of phosphosites induced by hypoxia, and a subset of cell line specific phosphorylation events [ 108 ].…”

“…As discussed, changes in phosphorylation states play key roles in regulating HIF signalling; however, large-scale changes in the phosphoproteome are observed in cells exposed to hypoxic stress [ 107 , 108 ]. A recent phosphoproteomic study analysing the hypoxia-induced phosphoproteome from four distinct melanoma cell lines revealed both a core set of phosphosites induced by hypoxia, and a subset of cell line specific phosphorylation events [ 108 ]. This analysis revealed that the mitogen-activated protein kinases (ERK1 and ERK2) and casein kinase were predicted to be active, with phosphorylation sites on the kinases differentially phosphorylated, and hypoxia-induced hyperphosphorylation of several predicted substrates [ 108 ].…”

“…A recent phosphoproteomic study analysing the hypoxia-induced phosphoproteome from four distinct melanoma cell lines revealed both a core set of phosphosites induced by hypoxia, and a subset of cell line specific phosphorylation events [ 108 ]. This analysis revealed that the mitogen-activated protein kinases (ERK1 and ERK2) and casein kinase were predicted to be active, with phosphorylation sites on the kinases differentially phosphorylated, and hypoxia-induced hyperphosphorylation of several predicted substrates [ 108 ]. These results indicate that hypoxia-induced signalling pathways are not only mediated by the PHD/VHL/HIF pathway.…”

Oxygen‐regulated post‐translation modifications as master signalling pathway in cells

“…If this data is extrapolated to consider the 72,000 high confidence Ser/Thr/Tyr phosphosites identified in the human proteome (Kalyuzhnyy et al , 2022), there may be as many as 10,000 hypoxia‐induced changes to the phosphoproteome. The limited hypoxia‐dependent phosphoproteomic experiments performed to date have revealed changes in phosphorylation of proteins in pathways as diverse as mitogen‐activated protein kinase pathways (ERK1 and ERK2), regulation of TGF (tumour growth factor)‐β receptor signalling, changes to Rho GTPase signalling and key factors involved in cytoskeletal organisation (Nilsson et al , 2010; Datta et al , 2021; Luo et al , 2022). These suggest that multiple kinase/phosphorylation‐dependent signalling pathways exist in tandem to modulate the cellular response in low oxygen, to tailor the response to different cellular contexts.…”