Dimethyl-2-ketoglutarate Stabilizes Hypoxia-inducible Factor-1α To Induce Highly Tumorigenic, Stem-like Properties In Breast Cancer Cells

Objective:To study the mechanism of dimethyl-2-ketoglutarate(DM-2KG, α-ketoglutarate analogues) upregulating hypoxia-inducible factor 1α(HIF-1α) and inducing breast cancer stem cells. Methods:Cell culture, including colony formation assay, trypan blue dye test, adenosine triphosphate(ATP) quantitative tests, breast cells ball formation assay, and dual luciferase reporter gene assay, Western analysis, small interfering RNA(si RNA) transfection, real-time quantitative polymerase chain reaction(q RT-PCR), semi-quantitative nested RT-PCR, senescence-associated β-galactosidase(SA-β-Gal) staining, immunofluorescence staining flow cytometry, and in vivo tumor xenograft animal experiments were used in this study. Results:1. α-ketoglutarate(α-KG) functions as a cofactor of prolyl hydroxylases(PHDs) to promote HIF-1α degradation. Unlike octyl-2KG,Dimethyl-2-ketoglutarate(DM-2KG), a cell membrane-permeable α-KG analogues, induced the expression of HIF-1α and further activated downstream target genes, such as GLUT1 and PDK1.2. DM-2KG transiently stabilized HIF-1α by inhibiting its degradation.3. DM-2KG promoted HIF-1α accumulation by inhibiting PHD2 mediated HIF-1αPro564 hydroxylation.4. DM-2KG,impeded breast cancer cell proliferation by inducing p21 expression and promoted quiescence.5. A cancer stem cell marker, CD44, was induced by prolonged DM-2KG treatment, resulting in a stem cell-like phenotype.6. Pre-treated MDA-MB-231 cells with DM-2KG stimulated tumorigenesis in a xenograft model.7. Knocking down HIF-1α abolished DM-2KG induction of the quiescent, cancer stem-like cells and the increased tumorigenic properties, confirming both these events were mediated through HIF-1α. Conclusion:DM-2KG induces HIF-1α by inhibiting PHD2,and then inhibits BC cell proliferation, and induces a subpopulation of BC cells to convert to CD44 high, Ki67 low quiescent, cancer stem-like cells that are highly tumorigenic. Therefore, targeting the PHD2-HIF-1α pathway has the unique potential to link metabolism to BC therapy resistance and disease progression, which is attributed to the cancer stem-like phenotype.DM-2KG may be a promising agent in cancer stem cell research.