Hexokinase 2 and nuclear factor erythroid 2-related factor 2 transcriptionally coactivate xanthine oxidoreductase expression in stressed glioma cells

is known to drive tumor progression. A considerable overlap between these processes exists, but several of their key regulators remain unknown. To this end, here we investigated the role of the proinflammatory cytokine IL-1β in connecting these processes in glioma cells. We found that glucose starvation sensitizes glioma cells to IL-1β-induced apoptosis in a manner that depended on reactive oxygen species (ROS). Although IL-1β-induced JNK had no effect on cell viability under glucose deprivation, it mediated nuclear translocation of hexokinase 2 (HK2). This event was accompanied by increases in the levels of sirtuin 6 (SIRT6), nuclear factor erythroid 2-related factor 2 (Nrf2), and xanthine oxidoreductase (XOR). SIRT6 not only induced (i) ROS-mediated cell death, but also facilitated (ii) a nuclear Nrf2–HK2 interaction, and (iii) recruitment of the Nrf2– HK2 complex to the ARE site on the XOR promoter. Importantly, HK2 served as transcriptional coactivator of Nrf2 to regulate XOR expression, indicated by decreased XOR levels in siRNA-mediated Nrf2 and HK2 knockdown experiments. Our results highlight a non-metabolic role of HK2 as transcriptional coactivator of Nrf2 to regulate XOR expression under conditions of proinflammatory and metabolic stresses. Our insights also underscore the importance of nuclear activities of HK2 in the regulation of genes involved in redox homeostasis.


Introduction
Emerging data indicate that interplay between inflammation and metabolism plays a critical role in tumor progression. In addition to its ability to extensively metabolise glucose for aiding increased energy demands, cancer cells are also under oxidative stress associated with increased production of ROS (1). The rapid glycolytic rate in glioblastoma (2) is concomitant with elevated levels of hexokinase-2 (HK2), that catalyzes the first step of the glycolytic pathway (3). While HK1 is the predominant isoform in low-grade gliomas, highly upregulated HK2 levels in GBM correlates with poor prognosis (3). The subcellular localization of HK2 is sensitive to extracellular glucose, with the distribution of HK2 between cytoplasm and mitochondria being dynamically regulated by glucose availability (4). HK2 regulates ROS levels (5), and glucose withdrawal increases ROS production in glioma cells (6). Also, the ability of diverse chemotherapeutic agents to induce glioma cell apoptosis through increased intracellular ROS generation is known (7)(8)(9).
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor which provides cytoprotection against oxidative stress. Oxidative stress mediates activation of Nrf2 (10) which is known to regulate ROS production by mitochondria and NADPH oxidase (11). In addition to contributing towards the maintenance of redox homeostasis, Nrf2 affects the expression of metabolic genes (12,13). Interestingly, SIRT6 not only regulates redox homeostasis by serving as an Nrf2 coactivator (14) but also affects glucose by guest on  http://www.jbc.org/ Downloaded from homeostasis via HIF-1α (15). Nrf2 regulates HIF-1α accumulation (16), and the latter serves as a regulator of HK2 (17). Moreover, IL-1 induced HK2 in glioma is dependent on relative abundance of HIF-1α-dependent SIRT6 levels (18). Also, HIF-1α-dependent subcellular localization of HK2 regulates cytoskeletal organization to consequently affect MHC-I clustering under inflammatory conditions in glioma cells (19).
Disrupting glycolytic flux serves as a trigger for inflammation and cell death (20).

Interestingly, glycolytic inhibitors and metabolic
conditions that affect hexokinase function and localization induce inflammasome activation involved in IL-1β secretion (21). Xanthine oxidoreductase (XOR), involved in catalyzing purines to uric acid, regulates IL-1 secretion upon NLRP3 inflammasome activation (22). Moreover, XOR is also known to regulate HIF-1through ROS in glioma cells (23). Given that inflammation rewires energy metabolism in the tumor microenvironment (24), the prospect of targeting altered metabolism in inflammation has been suggested as a substantial therapeutic promise (25). As there is a link between inflammation, metabolic status, and redox homeostasis, we investigated the effect of inflammation and glucose deprivation on oxidative stress in glioma cells.

IL-1-treated glioma cells is ROS-dependent
While treatment with IL-1or glucose deprivation alone had no effect on glioma cell viability, IL-1triggered a significant decrease in cell viability under low glucose conditions within 3 hours (Fig. 1a). Death was accompanied by alteration in the expression of molecules associated with cell cycle progression ( Supplementary Fig. 1a), and increase in cytochrome c and Bcl2 levels ( Supplementary   Fig. 1b). Cancer cells exhibit glucose metabolism-dependent inhibition of cytochrome c-mediated apoptosis (26), and glucose deprivation promotes ROS generation and death (6). An increase in ROS generation was observed in glucose-deprived cells treated with IL-1as compared to those exposed to low glucose condition (Fig 1b). As elevated ROS level induces glioma cell apoptosis upon inhibition of glucose metabolism (7) 2f).

SIRT6 regulates Nrf2
SIRT6 regulates oxidative stress responses by serving as an Nrf2 coactivator (14) and Nrf2 accumulation in cancer cells offers protection against oxidative stress (31). Importantly, Nrf2 is translocated to the nucleus in response to prooxidant stimuli (32

HK2 interacts with Nrf2 in a SIRT6dependent manner
As increased nuclear Nrf2 level was concomitant with increased nuclear HK2 accumulation, we investigated the association between the two.
Immunoprecipitation revealed increased association between Nrf2 and HK2 under glucose deprived conditions only in the presence of IL-1 (Fig 4c). Moreover, not only did siRNA-mediated SIRT6 knockdown decrease nuclear Nrf2 level in glucose deprived IL-1 treated cells, but it also abrogated Nrf2-HK2 interaction (Fig. 4c). These results provide strong evidence that Nrf2 is an interacting partner of HK2, and that SIRT6 is crucial in facilitating the interaction.

XOR is a target of HK2 and Nrf2
Nuclear translocation of Nrf2 in response to oxidative stress triggers transcriptional program through its binding to antioxidant response element (ARE) of antioxidant genes associated with maintenance of cellular redox balance (33).
HK2 serves as an intracellular glucose sensor of yeast cells to affect gene regulation (34) Fig. 2e).
Thus, the ability of HK2-Nrf2 complex to regulate Nrf2-dependent genes was found to be target specific and not all Nrf2 target genes follow HK2-dependent regulation.

NRF2 complex to ARE site on XOR promoter
Several studies have reported nuclear shuttling of HK2 (36,37), and in the context of SUC2 promoter HK2 functions as a transcriptional repressor (34). In view of our observations that XOR expression is regulated by both Nrf2 and HK2, the occupancy of Nrf2-HK2 complex at ARE site of XOR promoter to affect its expression was investigated. ChIP assay revealed increased enrichment of Nrf2 (Fig. 5d) as well as HK2 (Fig. 5e) (Fig. 5g).  luminometer (Promega), as described previously (45).

Confocal microscopy
For immunofluorescence staining, cells were grown in 4 well chamber glass slide system

Co-immunoprecipitation
Endogenous HK2 was immunoprecipated with anti-HK2 antibody from nuclear extracts obtained from treated and/or transfected cells. input was also resolved. Western blot analysis was performed with the immunoprecipitates and inputs with specific antibodies.

Xanthine oxidoreductase activity assay
The Amplex® Red Xanthine/Xanthine Oxidase Assay Kit (cat# a22182) was used for detecting xanthine oxidase activity in the cell lysates according to the manufacturer's instructions.

Chromatin immunoprecipitation (ChIP) and
ChIP-qPCR assay results were plotted as fold change over control.
All samples were normalised with their respective 18S rRNA CT values. qRT-PCR primers used were as follows:

Statistical Analysis
All comparisons between groups were performed either by two-tailed paired Students ttest or one-way ANOVA (Bonferroni's Multiple Comparison Test) for multiple comparisons between more than two groups. All p values of less than 0.05 were taken as significant.