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Transketolase Counteracts Oxidative Stress to Drive Cancer Development

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Transketolase Counteracts Oxidative Stress to Drive Cancer Development Transketolase counteracts oxidative stress to drive PNAS PLUS cancer development Iris Ming-Jing Xua, Robin Kit-Ho Laia,Shu-HaiLinb,c, Aki Pui-Wah Tsea, David Kung-Chun Chiua, Hui-Yu Koha, Cheuk-Ting Lawa, Chun-Ming Wonga,d, Zongwei Caib,c, Carmen Chak-Lui Wonga,d,1,andIreneOi-LinNga,d,1 aDepartment of Pathology, The University of Hong Kong, Hong Kong, SAR, China; bDepartment of Chemistry,Hong Kong Baptist University, Hong Kong, SAR, China; cState Key Laboratory of Environmental and Biological Analysis, Hong Kong Baptist University, Hong Kong, SAR, China; and dState Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, SAR, China Edited by Tak W. Mak, The Campbell Family Institute for Breast Cancer Research at Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada, and approved December 24, 2015 (received for review May 5, 2015) Cancer cells experience an increase in oxidative stress. The pentose When cells are in need of nucleotides, the PPP produces ribose phosphate pathway (PPP) is a major biochemical pathway that through the oxidative arm from G6P and the nonoxidative arm generates antioxidant NADPH. Here, we show that transketolase from F6P and G3P. (TKT), an enzyme in the PPP, is required for cancer growth because Although the PPP and glycolysis are equally important in the of its ability to affect the production of NAPDH to counteract metabolism of cells, attention has been mostly drawn to the oxidative stress. We show that TKT expression is tightly regulated mechanisms by which glycolysis benefits tumor growth. Knowledge by the Nuclear Factor, Erythroid 2-Like 2 (NRF2)/Kelch-Like ECH- regarding the roles of the PPP in cancer cells is relatively scarce. Associated Protein 1 (KEAP1)/BTB and CNC Homolog 1 (BACH1) Among all of the enzymes in the PPP, only the roles of G6PD and oxidative stress sensor pathway in cancers. Disturbing the TKTL1 were briefly revealed in cancer. G6PD and TKTL1 were redox homeostasis of cancer cells by genetic knockdown or reported to be activated or overexpressed in cervical, lung, gastric, pharmacologic inhibition of TKT sensitizes cancer cells to colorectal, and endometrial cancers (2–7). Suppressing TKTL1 in existing targeted therapy (Sorafenib). Our study strengthens the colorectal tumor cells reduced glucose uptake and lactate accu- notion that antioxidants are beneficial to cancer growth and mulation and enhanced sensitivity to oxidative stress (8). highlights the therapeutic benefits of targeting pathways that Hepatocellular carcinoma (HCC) is the most common type of generate antioxidants. primary liver cancer. It is the fifth most common cancer and the second leading cause of cancer deaths. The 5-y survival rate of TKT | HCC | ROS | metabolism | PPP HCC patients is less than 10% (9). Its high mortality rate is at- tributable to late symptom presentation and lack of promising etabolic reprogramming has recently been recognized as a curative therapy. Liver transplantation and tumor resection are so Mhallmark of cancer (1). Cancer cells preferentially use glycol- far the most effective HCC treatment. However, most HCC pa- ysis instead of oxidative phosphorylation to generate energy even in tients are not amenable to surgical treatments due to poor liver the presence of oxygen (O2). This metabolic shift, named the War- function or the presence of metastasis. Sorafenib, an oral multi- burg Effect, channels glucose intermediates for macromolecule and kinase inhibitor, is the only FDA-approved drug and extends the antioxidant synthesis. A very important metabolic pathway that connects with glycolysis is the pentose phosphate pathway (PPP). The Significance 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 Excessive accumulation of oxidative stress is harmful to cancer the two major redox molecules, glutathione and thioredoxin, in the cells. Our study demonstrates the important roles of a pentose reduced state. NADPH therefore counteracts reactive oxygen species phosphate pathway (PPP) enzyme, transketolase (TKT), in re- (ROS), enabling cancer cells to survive oxidative stress. dox homeostasis in cancer development. We highlight the The PPP is composed of the oxidative and nonoxidative arms. clinical relevance of TKT expression in cancers. We also show The oxidative arm of the PPP produces NADPH and ribose by that TKT overexpression in cancer cells is a response of Nuclear three irreversible steps. First, glucose-6-phosphate dehydrogenase Factor, Erythroid 2-Like 2 (NRF2) activation, a sensor to cellular (G6PD) converts glucose-6-phosphate (G6P) to 6-phospho-glu- oxidative stress. TKT locates at an important position that conolactone and NAPDH. Second, phosphogluconolactonase con- connects PPP with glycolysis to affect production of antioxi- dant NADPH. Our preclinical study shows that targeting TKT verts 6-phospho-gluconolactone to 6-phosphogluconate. Third, leads to elevation of oxidative stress, making cancer cells more 6-phosphogluconate dehydrogenase converts 6-phosphogluconate vulnerable to therapeutic treatment, such as Sorafenib. Using to ribulose-5-phosphate (Ru5P) and NAPDH. Ru5P then enters TKT as an example, our study suggests that targeting enzymes the nonoxidative arm of the PPP. Ru5P is converted to xylulose-5- for antioxidant production represents a direction for cancer phosphate (X5P) and Ru5P by epimerase and isomerase, re- treatment. spectively. The transketolase (TKT) family [transketolase-like 1 (TKTL1) and TKTL2] transfers two-carbon groups from X5P to Author contributions: I.M.-J.X., C.C.-L.W., and I.O.-L.N. designed research; I.M.-J.X., R.K.-H.L., R5P to generate sedoheptulose-7-phosphate (S7P) to glyceral- S.-H.L., A.P.-W.T., D.K.-C.C., H.-Y.K., C.-T.L., C.-M.W., and C.C.-L.W. performed research; I.M.-J.X., dehyde-3-phosphate (G3P). Transaldolase (TALDO) transfers S.-H.L., C.-T.L., C.-M.W., Z.C., C.C.-L.W., and I.O.-L.N. contributed new reagents/analytic tools; I.M.-J.X., S.-H.L., Z.C., C.C.-L.W., and I.O.-L.N. analyzed data; and I.M.-J.X., C.C.-L.W., and I.O.-L.N. three-carbon groups from S7P to G3P to generate erythrose-4- wrote the paper. phosphate (E4P) and fructose-6-phosphate (F6P). Finally, TKT The authors declare no conflict of interest. transfers two-carbon groups from X5P to E4P to generate G3P and This article is a PNAS Direct Submission. F6P, which reenter glycolysis. All enzymes in the nonoxidative arm Freely available online through the PNAS open access option. of the PPP are reversible, allowing cells to adapt to the dynamic 1To whom correspondence may be addressed. Email: [email protected] or carmencl@pathology. metabolic demands. When cells experience high oxidative stress, hku.hk. metabolites from the nonoxidative arm are rechanneled into gly- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. CELL BIOLOGY colysis to refill the oxidative arm for the synthesis of NAPDH. 1073/pnas.1508779113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1508779113 PNAS | Published online January 25, 2016 | E725–E734 Downloaded by guest on October 2, 2021 median survival of advanced HCC patients for about 3 mo. It is demonstrated that TKT was overexpressed by at least twofold in well known that HCC is one of the most rapidly growing solid 54.4% (56/103) of the HCC patients (Fig. 1C). Western blotting cancers. Furthermore, the liver is known to be a metabolic hub that further confirmed that TKT was also overexpressed at the protein is responsible for major metabolic events in the body such as blood level (Fig. 1D). Overexpression of TKT was also closely associated glucose homeostasis and glycogen metabolism. Nonetheless, the with aggressive clinicopathological HCC features, including the detailed metabolic alterations in HCC and the molecular mecha- presence of venous invasion and tumor microsatellite formation, nisms driving the metabolic adaptation in HCC remain largely increased tumor size, and absence of tumor encapsulation (Fig. 1E unknown. Better understanding of the metabolic machineries of and Table 2). Expression patterns of TKT and other PPP enzymes HCC will be beneficial for the development of therapeutic treat- from The Cancer Genome Atlas (TCGA) database containing ment. Here, using HCC as a cancer model, we demonstrate the transcriptome sequencing data of 50 paired HCC samples also imperative roles of the PPP in cancer development and illustrate an echo our own in-house data (Fig. S1A). Furthermore, the Onco- example that blocking a critical enzyme that connects glycolysis and mine database confirms that overexpression of TKT is not re- the PPP, TKT, would be sufficient to impede HCC development stricted to HCC but is also present in other cancer types such as and improve the efficiency of Sorafenib treatment in HCC. colorectal, bladder, gastric, ovarian, lung, renal, prostate, and breast cancers (Fig. S1B). Results PPP Enzymes Are Frequently Up-Regulated in Cancer. To gain insights NRF2 Competes with BACH1 to Activate TKT Expression. The next into the clinical implications of the PPP in cancer development, we questionweaskedwashowTKTwasup-regulatedincancer.A first compared the expression of all of the PPP enzymes in 16 pairs chromatin immunoprecipitation sequencing (ChIP-seq) study of human HCC and the corresponding nontumorous (NT) liver by revealed that a transcriptional repressor, BTB and CNC Ho- transcriptome sequencing (Table 1). Intriguingly, most PPP en- molog 1 (BACH1), can potentially bind to TKT (10). However, zymes were significantly up-regulated in human HCCs, suggesting the precise binding location has not been validated. BACH1 is that this pathway is deregulated in human HCC. Fragments per known to be an important regulator of oxidative stress through kilobase of transcript sequence per million mapped reads (FPKM) repressing heme oxygenase 1 (HMOX1), which degrades heme values suggested that TKT was the most abundantly expressed and to form bilirubin, a strong ROS scavenger (11).
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