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

SIMPLE SUMMARY: Most solid tumours, including melanoma (skin cancer), are riddled with areas lacking adequate oxygen supply due to insufficient vasculature. Cancer cells in these regions are resistant to therapies and contribute to cancer spread and poor treatment response in patients. Understanding the mechanisms by which cancer cells adapt to survive in such a hostile environment will provide novel avenues for treatment. In this study, we investigated mechanisms that melanoma cells use to adapt and survive in an oxygen-poor environment. We used four different melanoma cell lines and studied how protein levels and phosphorylation patterns on thousands of proteins change when the cells are exposed to poor oxygen conditions. This revealed potential mechanisms on which cancer cells are dependent for survival. These survival mechanisms can be potentially targeted to achieve durable response to therapy. We demonstrate this by targeting one such mechanism required for cancer cell survival. 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.