NECAB3 Promotes Activation of Hypoxia-inducible factor-1 during Normoxia and Enhances Tumourigenicity of Cancer Cells

Abstract

Unlike most cells, cancer cells activate hypoxia inducible factor-1 (HIF-1) to use glycolysis even at normal oxygen levels, or normoxia. Therefore, HIF-1 is an attractive target in cancer therapy. However, the regulation of HIF-1 during normoxia is not well characterised, although Mint3 was recently found to activate HIF-1 in cancer cells and macrophages by suppressing the HIF-1 inhibitor, factor inhibiting HIF-1 (FIH-1). In this study, we analysed Mint3-binding proteins to investigate the mechanism by which Mint3 regulates HIF-1. Yeast two-hybrid screening using Mint3 as bait identified N-terminal EF-hand calcium binding protein 3 (NECAB3) as a novel factor regulating HIF-1 activity via Mint3. NECAB3 bound to the phosphotyrosine-binding domain of Mint3, formed a ternary complex with Mint3 and FIH-1, and co-localised with Mint3 at the Golgi apparatus. Depletion of NECAB3 decreased the expression of HIF-1 target genes and reduced glycolysis in normoxic cancer cells. NECAB3 mutants that binds Mint3 but lacks an intact monooxygenase domain also inhibited HIF-1 activation. Inhibition of NECAB3 in cancer cells by either expressing shRNAs or generating a dominant negative mutant reduced tumourigenicity. Taken together, the data indicate that NECAB3 is a promising new target for cancer therapy.

Integrated functions of membrane‐type 1 matrix metalloproteinase in regulating cancer malignancy: Beyond a proteinase

“…(39) Mint3 is ubiquitously expressed, although some cancer cells express the protein at relatively higher levels. (53) However, Mint3 does not inhibit FIH-1 when MT1-MMP is absent. Genetic depletion of Mint3 (Mint3-KO) in mice does not exhibit severe phenotypes, except for macrophages that exhibit an ATP shortage, similarly to MT1-KO cells.…”

“…Mint3 is ubiquitously expressed, although some cancer cells express the protein at relatively higher levels . However, Mint3 does not inhibit FIH‐1 when MT1‐MMP is absent.…”

“…This finding indicates that MT1‐MMP‐mediated HIF activation is affected by the phosphoinositide 3‐kinase (PI3K)/Akt signaling pathway, which is usually activated in cancer cells . In addition, N‐terminal EF‐hand calcium binding protein 3 (NECAB3), which structurally resembles monooxygenase, binds to the PTB domain of Mint3 and promotes Mint3‐FIH‐1 interactions in cells . The monooxygenase activity of NECAB3 is presumably necessary for this function, but the precise mechanism remains unclear.…”

“…(56) In addition, N-terminal EF-hand calcium binding protein 3 (NECAB3), which structurally resembles monooxygenase, binds to the PTB domain of Mint3 and promotes Mint3-FIH-1 interactions in cells. (53) The monooxygenase activity of NECAB3 is presumably necessary for this function, but the precise mechanism remains unclear. Thus, further studies are needed to clarify the mechanism whereby NECAB3 promotes Mint3-FIH-1 interaction in cooperation with oncogenic signals.…”

Mint3/Apba3 depletion ameliorates severe murine influenza pneumonia and macrophage cytokine production in response to the influenza virus

“…We have recently reported that the monooxygenase NECAB3 and mTOR signalling also promote Mint3-mediated HIF-1 activation in cancer cells4445. Although whether NECAB3 relates to inflammation is unclear, mTOR contributes to inflammatory responses4647.…”