The transcription factor p73 triggers developmental pathways and overlaps stress-induced p53 transcriptional pathways. How p53-family response elements determine and regulate transcriptional specificity remains an unsolved problem. In this work, we have determined the first crystal structures of p73 DNA-binding domain tetramer bound to response elements with spacers of different length. The structure and function of the adaptable tetramer are determined by the distance between two half-sites. The structures with zero and one base-pair spacers show compact p73 DNA-binding domain tetramers with large tetramerization interfaces; a two base-pair spacer results in DNA unwinding and a smaller tetramerization interface, whereas a four base-pair spacer hinders tetramerization. Functionally, p73 is more sensitive to spacer length than p53, with one base-pair spacer reducing 90% of transactivation activity and longer spacers reducing transactivation to basal levels. Our results establish the quaternary structure of the p73 DNA-binding domain required as a scaffold to promote transactivation.T he p73 transcription factor that belongs to the p53 protein family and participates in pheromonal sensory, chromosome stability, neurogenesis, inflammation, and osteoblastic differentiation pathways (1, 2). In contrast to p53, p73 is mutated in less than 0.5% of human tumors (3); however, it also participates in p53-dependent and independent pathways, showing oncogenic and tumor suppressor functions (4, 5). These dual opposite activities are due to the presence of two promoters which results in the expression of two main isoforms, TAp73 and ΔNp73 (6).How the members of the p53 protein family trigger different cellular responses still remains an open question. Overall, p73 and p63 can bind to the same p53 response elements (REs), but the activated pathways are different (7,8). There is some redundancy in the activation of stress pathways by the three members of the p53 protein family, but, at the same time, over 100 genes regulated by p73 and p63 are not activated by p53 (9, 10). Like p53, p73 also binds to a 20-bp RE, comprising two half-site decamers in direct orientation that follow a 5′-Pur1-Pur2-Pur3-Cyt4-Ade5/Thy5-Ade6/Thy6-Gua7-Pyr8-Pyr9-Pyr10-3′ consensus sequence (10, 11). Half of the known p53 REs do not have any insertion between the two half-sites and spacers larger than 3 bp are rare, particularly among sites that are transcriptionally activated (12-14). In the case of p53 repressed genes, the cis-element code is poorly defined, but based on a limited number of examples, spacer length appears to be more uniformly distributed and targets have no preference for 0-bp spacers (12).Human p73α is a 636 amino acid protein with a tripartite domain organization similar to its close homolog, p63, and to the shorter 393 amino acid long p53 protein. Members of the p53 family have a disordered N-terminal transactivation domain, a central immunoglobulin-like DNA-binding domain (DBD), and a C terminus that starts with a domain that prom...

Background: Liver is well characterized as a major metabolic organ, the metabolic machineries driving liver cancer (hepatocellular carcinoma, HCC) progression remains poorly understood. Oxygen deprivation, hypoxia, is frequently found in regions of tumors with insufficient blood supply. Hypoxia stabilizes hypoxia-inducible factor-1α (HIF-1α) which transcriptionally activates genes that utilize glycolysis over oxidative phosphorylation for ATP production. This metabolic switch reduces mitochondrial reactive oxygen species (ROS) production that would otherwise occur due to electron imbalance through the electron transport chain (ETC) in hypoxic condition. In our transcriptome sequence analysis, among all the mitochondrial subunits in the complex I of the electron transport chain (ETC), only NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2 gene (NDUFA4L2) was distinctly and significantly induced by hypoxia and over-expressed in human HCC. However, the clinical implications and functions of this gene in HCC progression remain unknown. Methods: The mRNA expression of NDUFA4L2 in an expanded cohort of 100 HCC patients was evaluated by qRT-PCR. ChIP assay was performed to study the interaction of HIF-1α with NDUFA4L2. Stable knockdown by shRNA or knockout by TALEN of HIF-1α/NDUFA4L2 was established in HCC cells to explore the roles of NDUFA4L2 in HCC. ROS, mitochondrial membrane potential, and oxygen consumption rate were measured in the HIF-1α/NDUFA4L2 knockdown or knockout HCC cells. Orthotopic implantation model was employed to evaluate the in vivo effect of NDUFA4L2 and the efficiency of HIF inhibitors. Results: NDUFA4L2 was significantly overexpressed in human HCC and was markedly induced by hypoxia. Overexpression of NDUFA4L2 in human HCC was significantly correlated with aggressive HCC clinicopathological parameters including absence of tumor encapsulation, formation of tumor microsatellite, and poorer overall survival. ChIP assay confirmed the binding of HIF-1α with hypoxia response elements (HREs) in NDUFA4L2 promoter and expression study showed that hypoxia-induced NDUFA4L2 was abolished in HIF-1αknockdown or knockout HCC cells. Genetic ablation of HIF-1α/NDUFA4L2 increased the mitochondrial activity that coupled with higher intracellular reactive oxygen species (ROS) and ROS-induced apoptosis. Knockdown of NDUFA4L2 markedly suppressed tumor growth in vitro and in vivo. HIF inhibitors, digoxin and Sorafenib, profoundly inhibited the growth of HCC that expressed high levels of NDUFA4L2. Conclusion: Over-expression of NDUFA4L2 is associated with poor clinical outcome in HCC patients. Hypoxia-induced NDUFA4L2 reduced the mitochondrial ROS production and ROS-triggered apoptosis, thereby conferring HCC cells growth advantage in hypoxic environment. Targeting HIF-1α/NDUFA4L2 pathway by HIF inhibitors represent a new therapeutic strategy for HCC. Citation Format: Kit Ho Lai, Ming Jing Xu, Pui Wah Tse, Kung Chun Chiu, Wei Lai, Chun Ming Wong, Pik Wong, Oi Lin Ng, Chak Lui Wong. HIF-1α/NDUFA4L2 promotes hepatocellular carcinoma progression through reducing oxidative stress. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1188. doi:10.1158/1538-7445.AM2015-1188

Background and objectives: Hepatocellular carcinoma (HCC) is the primary liver malignancy with an extremely low survival rate. HCC progression is frequently associated with accelerated glucose consumption that confers growth advantage to tumor cell through two essential metabolic pathways - glycolysis and the pentose phosphate pathway (PPP). Glycolysis utilizes glucose for ATP production, while the PPP converts glycolytic intermediates to generate NADPH, the cellular antioxidant, and ribose-5-phophate, the nucleotide precursor. Although glycolysis has been extensively studied in HCC, how PPP supports HCC growth remains largely unknown. Our study aims at delineating the clinical significance, regulation and functions of PPP in HCC development and explore the therapeutic potential of PPP inhibitors for HCC therapy. Methods: The expression and abundance of the the PPP genes were examined in 16 pairs of human HCC tumors and adjacent non-tumors by transcriptome sequencing. Level of reactive oxygen species (ROS) and glucose uptake were measured by CM-H2DCFDA and 2-NBDG stainings, respectively. Metabolomics study was performed with CE-TOF-MS analysis. Metabolic flux analysis was conducted using UPLC-MS/MS. Results: Transcriptome sequencing data showed that enzymes in the PPP were frequently upregulated in HCC. Transketolase (TKT), the gene encodes the reversible enzyme connecting PPP and glycolysis, is the most abundant and most overexpressed PPP gene in HCC. Overexpression of TKT was confirmed in an expanded sample cohort at the mRNA and protein level. Meanwhile, TKT overexpression was significantly correlated with venous invasion, microsatellite formation, tumor size and absence of tumor encapsulation. Notably, TKT expression was controlled by NRF2/KEAP1/BACH1 pathway, which is a major transcription regulator for redox homeostasis. CHIP assay revealed that NRF2 and BACH1 competitively bound to the same antioxidant responsive elements (ARE) in TKT. Functionally, knockdown of TKT in HCC cells retarded cell growth, attenuated glucose uptake and NADPH production, increased intracellular ROS, and induced oxidative stress-associated cell cycle delay. In line with these findings, knockdown of TKT greatly suppressed tumor growth in vivo. Metabolomics and metabolic flux analysis revealed that loss of TKT disrupted the PPP and subsequently reduced NADPH production. Intriguingly, genetic knockdown and pharmacological inhibition of TKT enhanced the efficacy of Sorafenib, the only FDA-approved drug for HCC treatment, both in vitro and in vivo. Conclusion: Our study suggested the clinical significance of TKT in HCC and illustrated the anti-oxidative role of TKT in HCC progression. We also proposed that disrupting the metabolic machinery by TKT inhibition might be a novel therapeutic strategy for HCC treatment. Citation Format: Ming Jing Xu, Kit Ho Lai, Shu Hai Lin, Pui Wah Tse, David Kung Chun Chiu, Hui Yu Koh, Cheuk Ting Law, Chun Ming Wong, Zong Wei Cai, Carmen Chak Lui Wong, Irene Oi Lin Ng. Targeting pentose phosphate pathway (PPP) represents a novel therapeutic strategy for hepatocellular carcinoma (HCC) treatment. [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 1058.