Poor prognosis of cancers, including hepatocellular carcinoma (HCC), is mainly associated with metastasis; however, the underlying mechanisms remain poorly understood. This article investigates the role of lysyl oxidase-like 2 (LOXL-2) in the biology of HCC metastasis. First, we showed that HCC metastasis relies on a collagen-modifying enzyme, LOXL2, which was significantly overexpressed in tumorous tissues and sera of HCC patients, indicating that LOXL2 may be a good diagnostic marker for HCC patients. Second, we delineated a complex, interlinked signaling network that involves multiple regulators, including hypoxia, transforming growth factor beta (TGF-b), and microRNAs (miRNAs), converging to control the expression of LOXL2. We found not only that LOXL2 was regulated by hypoxia/hypoxia-inducible factor 1 alpha (HIF-1a), but also that TGF-b activated LOXL2 transcription through mothers against decapentaplegic homolog 4 (Smad4), whereas two frequently underexpressed miRNA families, miR-26 and miR-29, cooperatively suppressed LOXL2 transcription through interacting with the 3' untranslated region of LOXL2. Third, we demonstrated the imperative roles of LOXL2 in modifying the extracellular matrix components in the tumor microenvironment and metastatic niche of HCC. LOXL2 promoted intrahepatic metastasis by increasing tissue stiffness, thereby enhancing the cytoskeletal reorganization of HCC cells. Furthermore, LOXL2 facilitated extrahepatic metastasis by enhancing recruitment of bone-marrow-derived cells to the metastatic site. Conclusion: These findings integrate the clinical relevance, molecular regulation, and functional implications of LOXL2 in HCC metastasis. (HEPATOLOGY 2014;60:1645-1658 H epatocellular carcinoma (HCC) is the most common form of primary liver cancer. HCC is the fifth-most prevalent cancer and ranked the second-most common lethal cancer.1 It claims more than 600,000 lives globally every year. 1 The high mortality rate in HCC is mainly attributable to metastasis for at least three reasons. First, it is a major cause of liver and organ failure. Second, patients diagnosed

Cancer cells experience an increase in oxidative stress. The pentose phosphate pathway (PPP) is a major biochemical pathway that generates antioxidant NADPH. Here, we show that transketolase (TKT), an enzyme in the PPP, is required for cancer growth because of its ability to affect the production of NAPDH to counteract oxidative stress. We show that TKT expression is tightly regulated by the Nuclear Factor, Erythroid 2-Like 2 (NRF2)/Kelch-Like ECHAssociated Protein 1 (KEAP1)/BTB and CNC Homolog 1 (BACH1) oxidative stress sensor pathway in cancers. Disturbing the redox homeostasis of cancer cells by genetic knockdown or pharmacologic inhibition of TKT sensitizes cancer cells to existing targeted therapy (Sorafenib). Our study strengthens the notion that antioxidants are beneficial to cancer growth and highlights the therapeutic benefits of targeting pathways that generate antioxidants.M etabolic reprogramming has recently been recognized as a hallmark of cancer (1). Cancer cells preferentially use glycolysis instead of oxidative phosphorylation to generate energy even in the presence of oxygen (O 2 ). This metabolic shift, named the Warburg Effect, channels glucose intermediates for macromolecule and antioxidant synthesis. A very important metabolic pathway that connects with glycolysis is the pentose phosphate pathway (PPP). The major goal of the PPP is the production of ribose-5-phosphate (R5P) and NADPH. R5P is the major backbone of RNA and is critical to nucleotide synthesis. NADPH is the major antioxidant that maintains the two major redox molecules, glutathione and thioredoxin, in the reduced state. NADPH therefore counteracts reactive oxygen species (ROS), enabling cancer cells to survive oxidative stress.The PPP is composed of the oxidative and nonoxidative arms. The oxidative arm of the PPP produces NADPH and ribose by three irreversible steps. First, glucose-6-phosphate dehydrogenase (G6PD) converts glucose-6-phosphate (G6P) to 6-phospho-gluconolactone and NAPDH. Second, phosphogluconolactonase converts 6-phospho-gluconolactone to 6-phosphogluconate. Third, 6-phosphogluconate dehydrogenase converts 6-phosphogluconate to ribulose-5-phosphate (Ru5P) and NAPDH. Ru5P then enters the nonoxidative arm of the PPP. Ru5P is converted to xylulose-5-phosphate (X5P) and Ru5P by epimerase and isomerase, respectively. The transketolase (TKT) family [transketolase-like 1 (TKTL1) and TKTL2] transfers two-carbon groups from X5P to R5P to generate sedoheptulose-7-phosphate (S7P) to glyceraldehyde-3-phosphate (G3P). Transaldolase (TALDO) transfers three-carbon groups from S7P to G3P to generate erythrose-4-phosphate (E4P) and fructose-6-phosphate (F6P). Finally, TKT transfers two-carbon groups from X5P to E4P to generate G3P and F6P, which reenter glycolysis. All enzymes in the nonoxidative arm of the PPP are reversible, allowing cells to adapt to the dynamic metabolic demands. When cells experience high oxidative stress, metabolites from the nonoxidative arm are rechanneled into glycolysis to refill the oxidative arm for...

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer. It ranks third amongst cancer-related deaths worldwide owing to its late symptom presentation and lack of effective treatment regimens. Compared to normal cells, cancer cells experience distinctly higher amount of oxidative stress from increased reactive oxygen species (ROS) generation contributed by metabolic alterations, or its hypoxic microenvironment. Cancer cells also have a greater capacity of antioxidant production to maintain redox homeostasis. Immense generation of NADPH, a major metabolite and antioxidant is found in cancer cells to combat ROS as confirmed in our previous studies as we identified the folate cycle and pentose phosphate pathway (PPP) as major metabolic pathways responsible for NADPH production in human HCC. NADPH is an electron donor which can reduce and activate different antioxidant systems. The thioredoxin system is a ubiquitously-expressed mammalian antioxidant system activated by NADPH. The transmission of an electron from NADPH to TXN, the ROS-scavenging member of the thioredoxin system, is modulated by thioredoxin reductase 1 (TXNRD1). TXRND1 is the sole activating-enzyme of the thioredoxin system, playing a vital role in maintaining intracellular redox homeostasis. Furthermore, TXNRD1 was found to be significantly over-expressed in human HCC correlated with poor clinical prognosis and patient survival. Genetic inhibition of TXNRD1 via shRNA-knockdown significantly induced oxidative stress which suppressed HCC cell proliferation in vitro and liver tumor formation orthotopically implanted in vivo. Interestingly, TXNRD1-inhibition-induced oxidative stress rendered HCC cells more sensitive towards Sorafenib, its conventional therapeutic agent. This was highlighted by a dramatic induction of ROS-induced apoptosis in vitro and suppression tumor growth in vivo. NRF2, a transcription factor and master regulator against oxidative stress, was confirmed to bind with TXNRD1 using ChIP assay. Both genetic inhibition and pharmacological activation of NRF2 in HCC cells confirmed the dependent relationship between NRF2 and TXNRD1. Therapeutically, pharmacological inhibition of the thioredoxin system using the TXNRD1 inhibitor auranofin greatly sensitized HCC cells towards sorafenib. The synergism was observed through significant induction of ROS resulting in significant cell death in vitro and suppression of tumor formation in vivo. Our investigation demonstrated the induction of oxidative stress, as a result of thioredoxin system inhibition, rendered HCC cells more sensitive towards therapeutic treatment. Contrasting the widely-known, tumorigenic role of ROS, we now shed light on a paradigm-shifting strategy which ROS may be used as a therapeutic adjuvant to combat cancer. Citation Format: Derek Lee, Iris Ming Jing Xu, David Kung Chun Chiu, Robin Kit Ho Lai, Chun Ming Wong, Irene Oi Lin Ng, Carmen Chak Lui Wong. Oxidative stress induction through TXNRD1 inhibition as a therapeutic strategy in hepatocellular carcinoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2445.