Abstract 2998: MLN4924 inhibition of Cullin-Ring ligases activates a hypoxic response and secretion of tumor promoting factors in human MCF7 breast cancer cells
Authors: Rivard James , James Amy , Eleria Erin , Lee Neal , Brumbaugh Kathy , Wegner Greta , Reagan Kevin 
Published: August 2015

Hypoxia-inducible factors (HIFs) are the key transcription factors that mediate the cellular response to low oxygen tension. During normoxia, HIF activity is tightly controlled by its oxygen-dependent prolyl hydroxylation which allows polyubiquitination by von Hippel-Landau tumor suppressor (VHL), leading to HIF degradation by the ubiquitin-proteasome pathway. During hypoxia, low oxygen tension prevents HIF prolyl hydroxylation resulting in HIF accumulation and subsequent transcription of HIF target genes. VHL is a known Cullin-RING E3 ubiquitin ligase (CRL) that is dependent on neddylation of the Cul-2 subunit by NEDD8 Activating Enzyme (NAE) for its enzymatic activity. The small molecule inhibitor MLN4924 inhibits NAE neddylation of Cullins by covalently binding the nucleotide-binding site of NAE. HIF-1α is a well-known CRL substrate that increases in abundance upon MLN4924 treatment. We investigated the response of MCF-7 human breast cancer cells to MLN4924 treatment compared to hypoxia (1% O2, 48 hours) treatment. Cell lysates tested using the Human Oncology Antibody Array and Western blot showed that both treatments induced accumulation of HIF-1α protein, with protein levels in MLN4924 treated cells greater than levels in hypoxia treated cells. This correlated with the expected attenuation of HIF-1α ubiquitination as detected by the Human Ubiquitin Antibody Array. The specificity of MLN4924 targeting Cullin proteins was tested using Western blots. The neddylation of the VHL subunit Cul-2, as well as Cul-1, was not detectable in MLN4924 treated cells, but was readily detectable in hypoxia treated cells. Since MLN4924 was reported to inhibit mTORC1 activity, Western blots were used to evaluate signaling of the mTORC1 complex. Thr-389 phosphorylation of p70 S6K was not detectable upon treatment with MLN4924 and attenuated in hypoxia treated cells. Thr-389 phosphorylated p70 S6K was not detectable in Rapamycin-treated cells, a potent mTORC1 inhibitor. The relative protein levels of several known HIF-1α target genes were tested using the Human Angiogenesis Antibody Array. VEGF was detected in cell culture supernates from normoxia, hypoxia, and MLN4924 treated cells. ELISAs were used to measured VEGF and Carbonic Anhydrase IX and were found to be highest in MLN4924 treated cells. Interestingly, these same array profiles detected tumor promoting factors Angiogenin, Amphiregulin, Endothelin 1, and PDGF-AA in cell supernates from MLN4924 treated cells at higher levels than hypoxia treated MCF-7 cells. Recent reports found MLN4924 to inhibit tumor angiogenesis in mouse models, indicating that the effects of MLN4924 on secretion of tumor promoting factors by MCF-7 cells may be context dependent.

Citation Format: James Rivard, Amy James, Erin Eleria, Neal Lee, Kathy Brumbaugh, Greta Wegner, Kevin Reagan. MLN4924 inhibition of Cullin-Ring ligases activates a hypoxic response and secretion of tumor promoting factors in human MCF7 breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2998. doi:10.1158/1538-7445.AM2015-2998