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

A dynamic network of metabolic adaptations, inflammatory responses, and redox homeostasis is known to drive tumor progression. A considerable overlap among these processes exists, but several of their key regulators remain unknown. To this end, here we investigated the role of the proinflammatory cytokine IL-1ß in connecting these processes in glioma cells. We found that glucose starvation sensitizes glioma cells to IL-1ß-induced apoptosis in a manner that depended on reactive oxygen species (ROS). Although IL-1ß-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 ROS-mediated cell death but also facilitated nuclear Nrf2-HK2 interaction. Recruitment of the Nrf2-HK2 complex to the ARE site on XOR promoter regulated its expression. 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.

SIRT6 regulated nucleosomal occupancy affects Hexokinase 2 expression.

To understand the molecular association between inflammation and dysregulated metabolism in glioblastoma, the effect of IL-1ß on Hexokinase 2 (HK2) expression was investigated. IL-1ß induced HK2 expression was accompanied by heightened SIRT6 and MZF1 levels. IL-1ß mediated overall decrease in chromatin compactness on HK2 promoter involved diminished nucleosomal occupancy around the most labile region bearing MZF1 sites. Importantly, SIRT6 and MZF1 served as negative regulators of HK2. Ectopic SIRT6 induced formation and recruitment of MZF1-SIRT6 complex to MZF1 site was concomitant with increased nucleosomal occupancy. The function of SIRT6 as co-repressor of MZF1 was inconspicuous in cells treated with IL-1ß alone, as IL-1ß-induced HIF-1α prevented SIRT6 availability for interaction with MZF1. Taken together, SIRT6 over-expression establishes a condition whereby reconfiguration of the HK2 promoter chromatin structure makes it receptive to interaction with MZF1/SIRT6 complex, thereby favouring a regulatory state conducive to diminished transcription.

TNFα driven HIF-1α-hexokinase II axis regulates MHC-I cluster stability through actin cytoskeleton.

Hypoxia-inducible Factor-1α (HIF-1α)-regulated expression of Hexokinase-II (HKII) remains a cornerstone in the maintenance of high metabolic demands subserving various pro-tumor functions including immune evasion in gliomas. Since inflammation-induced HIF-1α regulates Major Histocompatibility Complex class I (MHC-I) gene expression, and as cytoskeletal dynamics affect MHC-I membrane clusters, we investigated the involvement of HIF-1α-HKII axis in Tumor Necrosis Factor-α (TNFα)-mediated MHC-I membrane cluster stability in glioma cells and the involvement of actin cytoskeleton in the process. TNFα increased the clustering and colocalization of MHC-I with cortical actin in a HIF-1α dependent manner. siRNA mediated knockdown of HIF-1α as well as enzymatic inhibition of HK II by Lonidamine, delocalized mitochondrially bound HKII. This altered subcellular HKII localization affected TNFα-induced cofilin activation and actin turnover, as pharmacological inhibition of HKII by Lonidamine decreased Actin-related protein 2 (ARP2)/cofilin interaction. Photobleaching studies revealed destabilization of TNFα- induced stable MHC-I membrane clusters in the presence of Lonidamine and ARP2 inhibitor CK666. This work highlights how TNFα triggers a previously unknown function of metabolic protein HKII to influence an immune related outcome. Our study establishes the importance of inflammation induced HIF-1α in integrating two crucial components- the metabolic and immune, through reorganization of cytoskeleton.

Lactate induced HIF-1α-PRMT1 cross talk affects MHC I expression in monocytes.

Tumor infiltrating monocytes play a crucial role in tumor immune surveillance. As lactate is an important component of the tumor milieu, we investigated its role in the transcriptional regulation of MHC I which is crucial for mounting effective immune responses against tumors. Lactate elevated MHC class I expression in monocytes. Increase in HLAB expression was concomitant with increase in HIF-1α and decrease in PRMT1 levels. Interestingly, a reciprocal relationship was observed between PRMT1 and HIF-1α. While HIF-1α inhibition decreased lactate induced MHC I, both pharmacological inhibition and siRNA mediated knockdown of PRMT1 upregulated HLAB levels. PRMT1 over-expression rescued lactate mediated increase in MHC I expression. Lactate mediated changes in nucleosomal occupancy on HLAB promoter facilitated a chromatin landscape that favoured decreased recruitment of CREB and PRMT1 on CRE site of HLAB locus. The effect of lactate on the chromatin landscape of HLAB was completely mimicked by PRMT1 inhibitor AMI-1 in terms of nucleosomal occupancy and CREB recruitment. Besides demonstrating the importance of lactate in the transcriptional regulation of HLAB, this study highlights for the first time the (i) existence of HIF-1α-PRMT1 regulatory loop and (ii) role of PRMT1 in modulating chromatin landscape crucial for facilitating HLAB gene expression.

Oncrasin targets the JNK-NF-κB axis to sensitize glioma cells to TNFα-induced apoptosis.

Resistance of glioblastoma multiforme (GBM) to tumor necrosis factor (TNF) α-induced apoptosis have been attributed to increased nuclear factor-kappaB (NF-κB) activation. As we have previously reported that certain anticancer chemotherapeutics can sensitize glioma cells to TNFα-induced apoptosis by abrogating NF-κB activation, we investigated the potential of oncrasin in sensitizing glioma cells to TNFα-induced apoptosis. Oncrasin reduced glioma cell viability, inhibited TNFα-mediated NF-κB activation and sensitized cells to TNFα-induced apoptosis. Apoptosis was accompanied by elevated Fas and Fas-associated death domain (FADD) levels, increased caspase-8 activation and formation of death-inducing signaling complex (DISC). Oncrasin also (i) affected expression of cell cycle regulators, (ii) triggered DNA damage response, (iii) induced G(2)/M cell cycle arrest, (iv) decreased telomerase activity, (v) abrogated STAT3 activation and (v) mediated extracellular release of high mobility group box 1 (HMGB1) along with its increased association with nucleosomes. Oncrasin-induced apoptosis did not involve mitochondria. Importantly, oncrasin increased c-jun N-terminal kinase (JNK) phosphorylation and pharmacological inhibition of JNK rescued oncrasin-induced apoptosis. JNK inhibition prevented oncrasin-induced decrease in TNFα-induced NF-κB activity and inhibition of NF-κB increased JNK phosphorylation in TNFα-treated cells. Oncrasin induced DISC formation and inhibited anchorage-independent growth of glioma cells in a JNK-dependent manner. By elucidating the existence of JNK-NF-κB cross-talk that regulates resistance to TNFα-induced apoptosis, this study has highlighted the importance of JNK in regulating viability of glioma cells.

ß-defensin-3 negatively regulates TLR4-HMGB1 axis mediated HLA-G expression in IL-1ß treated glioma cells.

The non-classical HLA class I antigen HLA-G contributes to immune escape mechanisms in glioblastoma multiforme (GBM). We have previously shown that IL-1ß-HIF-1α axis connects inflammatory and oncogenic pathways in GBM. In this study, we investigated the role of IL-1ß induced inflammation in regulating HLA-G expression. IL-1ß increased HLA-G and Toll like receptor 4 (TLR4) expression in a HIF-1α dependent manner. Inhibition of TLR4 signaling abrogated IL-1ß induced HLA-G. IL-1ß increased HMGB1 expression and its interaction with TLR4. Inhibition of HMGB1 inhibited TLR4 and vice versa suggesting the existence of HMGB1-TLR4 axis in glioma cells. Interestingly, HMGB1 inhibition prevented IL-1ß induced HLA-G expression. Elevated levels of HMGB1 and ß-defensin 3 were observed in GBM tumors. Importantly, ß-defensin-3 prevented IL-1ß induced HLA-G, TLR4, HMGB1 expression and release of pro-inflammatory mediators. Our studies indicate that ß-defensin-3 abrogates IL-1ß induced HLA-G expression by negatively affecting key molecules associated with its regulation.