Abstract
One of the requirements for tumor development is blood supply, most often driven by hypoxia-induced angiogenesis. Hypoxia induces the stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), which induces expression of an angiogenic factor, vascular endothelial growth factor (VEGF). The purpose of this study is to validate a new screening platform combined with orthogonal assays to rapidly identify HIF-1 inhibitors and to evaluate the effectiveness of approved drugs on modulating HIF-1 signaling.We generated an endogenous HIF-1α–NanoLuc luciferase reporter allele in the human HCT116 colon cancer cell line using genome editing and screened a panel of small interfering RNAs (siRNAs) to 960 druggable targets and approximately 2,500 drugs on a quantitative high-throughput screening (qHTS) platform. Selected compounds were further investigated with secondary assays to confirm their anti-HIF activity and to study their mode of action. The qHTS assay identified over 300 drugs that inhibited HIF-1α-NanoLuc expression. The siRNA screening results supported the effectiveness of several target-specific inhibitors. Moreover, the identified HIF-1 inhibitors, such as mycophenolate mofetil, niclosamide, and trametinib, were able to suppress cancer cell proliferation and angiogenesis. Our study indicates that blocking the mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K) pathways effectively inhibits hypoxia-induced HIF-1α accumulation and HIF-1α transactivation and that proteasome inhibitors induce accumulation and decrease transcriptional activity of HIF-1α. These findings underline the importance of developing a battery of robust assay platforms and confirmation studies that focus on endogenous protein targets so that only relevant and reliable data will be taken into pre-clinical and clinical studies.
Highlights
Hypoxia-inducible factors (HIFs) have a crucial role in cancer development, progression, and metastasis [1]
Www.impactjournals.com/oncotarget relative luminescence unit (RLU) values measured from the HIF- 1α-Nano Luciferase (NanoLuc) reporter were proportional to the HIF- 1α-NanoLuc protein levels measured by western blotting (Figure S1B)
Consistent with the findings from an independent study that reported an small interfering RNAs (siRNAs) screen in melanoma cells [9], our results revealed that silencing of the three DNA damage response (DDR) factors—ATM, checkpoint kinase 1 (CHEK1) and checkpoint kinase 2 (CHEK2)— led to altered HIF-1α-NanoLuc expression levels under hypoxic conditions and the three genes are involved in several signaling pathways and networks identified by the siRNA screen (Table S3 and S4)
Summary
Hypoxia-inducible factors (HIFs) have a crucial role in cancer development, progression, and metastasis [1]. Hypoxia results in the stabilization of the labile HIF-1 subunit HIF-1α and the induction of HIF-1 target gene transcription. The increased expression of HIF-1 target genes, including vascular endothelial growth factor (VEGF) and other angiogenic genes, results in blood vessel formation and tumor expansion. HIF-1α is a potential target for inhibition of both tumor-mediated angiogenesis and other aspects of tumor development, such as metabolic alterations that further increase the proliferation of tumor cells. Over 20 HIF- 1 inhibitors, including topotecan (Hycamtin), vorinostat (Zolinza) and YC-1, which are approved anti-cancer drugs, have been tested in clinical trials, or are being investigated in pre-clinical studies [3]
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