ADP-dependent glucokinase regulates epithelial-to-mesenchymal transition under hypoxia in cancer cells

The tumour microenvironment (TME), typically characterised by low O2 (hypoxia), scarce nutrients and low pH, drives cancer cells (CCs) into a metastatic and resistant phenotype primarily responsible for patient mortality. The role of the archaeal alternative glycolytic enzyme, ADP-dependent glucokinase (ADPGK), in CCs under 1% O2 remains elusive. This study aimed at closely mimicking the hypoxic TME conditions in vitro to assess the role of ADPGK on epithelial-to-mesenchymal transition (EMT) that leads to CC metastatic dissemination. Lentiviral-mediated shRNA-dependent ADPGK knockdown in A549 and HCT116 cells was used as the loss-of-function (loss-of-function) model. CCs were exposed to hypoxia (1% O2) under euglycaemia (5.5 mM glucose) or hypoglycaemia (2.0 mM glucose) for 48/72 h, using 20% O2 under standard culture conditions as a non-hypoxic control. Viable CC numbers and EMT signalling were measured with trypan blue and RT-qPCR-based assays, respectively. The transwell and scratch assays were carried out to examine directed and non-directed CC motility. Hypoxic CC cycle progression was measured via propidium-iodide staining and flow cytometry. The ability of CCs to survive in a low-density environment after hypoxia/hypoglycaemia was measured through clonogenic assays. The Cancer Genome Atlas (TCGA) transcriptomic data of the lung adenocarcinoma (LUAD) shows lower ADPGK mRNA in lung tumour samples. In contrast, the colon adenocarcinoma (COAD) data shows the opposite. The high ADPGK mRNA is associated with poor overall survival in colon cancer patients. ADPGK loss-of-function decreased the A549 growth curve in hypoglycaemia under 20% O2 and had a similar effect in HCT116 cells but under hypoglycaemia and 1% O2. The culture conditions mimicking TME reveal the role of ADPGK in modulating cell growth without affecting cell cycle progression in CCs. Low-density survival in A549 ADPGK deficient cells increased under 20%/1% O2, suggesting ADPGK suppress CC survivability. ADPGK upregulates EMT-associated genes CDH2, IGFBP3 and ZEB2, whilst SNAI2 mRNA was reduced in A549 cells under euglycaemia and 1% O2; and under similar conditions, ADPGK modulates CDH1 and PKP2 in HCT116 cells, indicating a cell-dependent ADPGK modulation of EMT. The ADPGK-mediated upregulation of EMT-associated genes promotes cell-directed migration in A549 cells under euglycaemic hypoxia. Despite the upregulation of EMT-promoting genes, ADPGK loss-of-function impaired the A549 non-directed cell migration in euglycaemia/hypoglycaemia under 1% O2, implying ADPGK regulates cell motility depending on the extracellular environment. ADPGK prevents excessive collective CC motility, migration, and cell growth that would otherwise lead to ATP depletion and killing of the CC. The active EMT state promotes single-cell migration towards the chemoattractant in ADPGK loss-of-function A549 cells, signalling the immediate need to scour energy sources to avoid cell death. Hypoxia increases ADPGK mRNA, which is in agreement with the ADPGK transcript level in the colon tumour, which is also hypoxic. Euglycaemia, coupled with serum starvation and hypoxia, reverses the diminished ADPGK mRNA in the ADPGK loss-of-function cells. ADPGK loss-of- function in HCT116 reduces cell growth and slightly diminishes cell directed under hypoglycaemic and euglycaemic hypoxia, respectively. Overall, our research shows that the protein ADPGK plays a crucial role in how cancer cells adapt to harsh environments like the tumour microenvironment (TME). ADPGK affects cancer cell growth, migration, and survival via modulation of EMT. Therefore, targeting ADPGK could make cancer cells more vulnerable to stresses, potentially leading to cell death. This provides an alternative approach to targeting hypoxic CCs that have acquired resistance to cancer treatments.