Identification of novel protein regulators of the HIF signaling pathway by genome-wide screening

Abstract

Cancer is a leading cause of death worldwide, with brain tumors and lung cancers demonstrating particularly high mortality rates. The role of the complex tumor microenvironment (TME) and the interactions of cancer cells with the tumor stroma are the focus of intense investigation to better understand cancer progression and to test potential therapeutic options. In many solid tumors, regions with reduced oxygen availability (hypoxia) are present in the TME, along with active hypoxia inducible factor (HIF) signaling, the main cellular oxygen-sensing pathway. While the core HIF pathway is well characterized, its regulation remains to be fully elucidated. Uncovering the regulation of HIF signaling is key to understand how brain and lung tumors develop, adapt to low or fluctuating oxygen tensions and hijack the HIF pathway for progression, e. g., to induce invasion and metastasis. To discover novel HIF pathway regulators, in vitro fluorescent reporters (FRs) were developed to monitor the activation status of this signaling pathway in cells. Selected FRs were further examined in glioblastoma and/or lung adenocarcinoma cell lines to generate fluorescent reporter systems (FRSs). The best-performing FRSs were used to optimize the experimental conditions for a CRISPR knock-out (KO) screen targeting the whole coding genome. This screening in a glioblastoma FRS identified several known and previously unknown candidate regulators of the HIF signaling pathway in normoxia. Selected top hits were independently validated in both glioblastoma and lung adenocarcinoma FRSs. Moreover, initial functional analyses on a top hit, suppressor of cytokine signaling 3 (SOCS3), were performed in several cancer cell lines, including additional glioblastoma as well as renal cell carcinoma cell lines, in order to uncover the role of SOCS3 in HIF regulation. The development and application of the FRSs have moved us closer to decoding the regulation of the HIF signaling pathway in multiple cancers. Going forward, we will follow up on these findings to gain mechanistic insights into how SOCS3 regulates the HIF pathway. In addition, the versatile FRSs will help us to identify additional regulators under varying tumor microenvironmental conditions, such as hypoxia. By uncovering the regulation of the HIF signaling pathway in more detail, these studies may contribute to the development of innovative therapeutic strategies, paving the way to more effectively combat cancer.