Abstract 2793: ADP- dependent glucokinase enhances hypoxia- inducible factor-α target gene transactivation through modulation of ROS levels in hypoxic human cancer cells
Authors: Rey Sergio , Schito Luana , Koritzinsky Marianne , Wouters Bradly G. 
Published: July 2016

ADP- dependent glucokinase (ADPGK) is a glycolytic enzyme catalyzing the reaction of glucose→glucose 6-P by using ADP instead of ATP. ADPGK is remarkably conserved across various phyla from archaea to humans. Despite an important role priming glycolysis in conditions of metabolic stress in extremophile organisms, human ADPGK does not contribute to glycolysis. Recent work shows that ADPGK is essential for ROS generation during T-cell activation. Intriguingly, ADPGK protein levels are upregulated across a panel of cancer cell lines and primary tumors. Hereby we hypothesized that the increase of cellular ROS in response to mild (∼1% O2) hypoxia is dependent on ADPGK therefore contributing to the stabilization of hypoxia- inducible factor (HIF)-1α, a central regulator of the transcriptional response of cancer cells to hypoxia.

We achieved ADPGK loss-of-function (LOF) utilizing lentiviral shRNA- mediated knockdown (&gt;75%) in HCT-116 and HT-29 (colon); or H460 (lung) cancer cell lines. Intracellular ROS levels under hypoxia (1% O2) were analyzed by CM-H2DCFDA fluorescence and flow cytometry. Protein levels of HIF-1α (and -2α), and selected HIF-α targets were assessed by immunoblot assays. HIF-1α (and -2α) transcriptional activity was measured through a RT-qPCR array developed in house targeting 84 hypoxia- responsive transcripts in HCT-116 cells exposed to 20, 10, 5, 1, .2 and &lt;.02% O2 for 24 h. Baseline non-hypoxic oxygen consumption and glycolytic activities were measured in real-time with an automated bioanalyzer (Seahorse Biosciences) whereas 3D spheroid growth assays and HCT-116 xenografts were used to assess the effect of ADPGK LOF on tumor mass.

ADPGK LOF decreased ROS and HIF-1α (-and 2α) protein levels in a cell type- and O2- dependent manner in HCT-116, HT-29 and H460 cells. Results from our RT-qPCR array show a blunted transcriptional response to hypoxia with maximal inhibition at .2% O2 (∼15 mmHg). Immunoblots confirmed a striking impairment of CA9 hypoxic induction in addition to a significant decrease of the HIF-α targets IGFBP3, ERO-1α and DDIT4. 3D spheroid assays showed a significant growth rate increase after ADPGK knockdown. Addition of the antioxidant N-Ac-Cysteine during the spheroid formation phase mimicked the effect of ADPGK LOF in control cells whereas it further enhanced cell growth in ADPGK knockdown. However, baseline O2 consumption and glycolysis were not affected. Consistently with results of 3D spheroid assays, we observed enhanced tumor growth of HCT-116 xenografts bearing ADPGK LOF.

Our work uncovers a hitherto unknown function of ADPGK in cancer cells whereby it enhances ROS- dependent HIF-1α (and -2α) hypoxic stabilization and transactivation therefore limiting tumor growth. Further work will be focused on the mitochondrial origin of ADPGK- dependent hypoxic ROS generation as suggested by previous studies in primary T-lymphocytes.

Citation Format: Sergio Rey, Luana Schito, Marianne Koritzinsky, Bradly G. Wouters. ADP- dependent glucokinase enhances hypoxia- inducible factor-α target gene transactivation through modulation of ROS levels in hypoxic human cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2793.