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 antioxidant 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). BACH1 binds most profoundly up-regulated PPP enzyme in HCC (Fig. 1A). Of to the antioxidant response elements (AREs) of HMOX1 (11). note, two other members in the TKT family, TKTL1 and TKTL2, Inspired from these findings, we performedinsilicoanalysis were barely detectable, suggesting that TKT is the predominant and found seven putative AREs in TKT with consensus nucle- form in the context of liver. We further confirmed our observation otides, “-TGACTC-” (10) (Fig. 2A and Fig. S2A). ChIP assay in an expanded cohort of 103 HCC cases (Fig. 1B). Waterfall plot was performed in a HCC cell line, MHCC97L, and we confirmed

Fig. 1. TKT was overexpressed in HCC samples, and overexpression correlated with aggressive clinicopathological features. (A) Transcriptome sequencing data showing the expression of TKT, TKTL1, and TKTL2 in 16 pairs of HCC and NT liver samples. Gene expression is represented as FPKM. (B) qRT-PCR analysis of mRNA levels of TKT in 103 pairs of HCC tumor and NT samples. HPRT was used as the internal control. (C) Waterfall plot shows that TKT was up-regulated in 54.4% (56/103) of human HCC sam-

ples by at least twofold. –ΔΔCT = –[(CTTKT – CTHPRT) of HCC – (CTTKT – CTHPRT) of NT]. (D) Protein expression of TKT in representative cases of HCC and their corresponding NT liver tissues was determined by Western blotting. Intensities of bands were analyzed by Image J and normalized to the corresponding NT samples. (E) TKT expression in HCC correlated with aggressive clinicopathological features of HCC, including venous invasion, tumor microsatellite formation, tumor size, and absence of tumor encapsulation. *P < 0.05, **P < 0.01, ***P < 0.001. A,pairedt test; B, Wilcoxon signed rank test; E,Mann–Whitney test.

E726 | www.pnas.org/cgi/doi/10.1073/pnas.1508779113 Xu et al. Downloaded by guest on October 2, 2021 Table 1. Expression of PPP genes in clinical HCC samples PNAS PLUS Gene Mean FPKM, HCC Mean FPKM, NT Fold change, HCC/NT P value, t test

TKT 101.722 22.797 4.462 0.0442 G6PD 7.807 2.080 3.753 0.0001 TKTL2 0.005 0.001 3.659 0.4375 RPE 7.805 3.959 1.971 0.0004 PGLS 37.050 19.745 1.876 0.0007 RPIA 6.216 3.377 1.84 0.00006 PGD 34.922 20.841 1.676 0.0495 TALDO1 93.148 61.181 1.522 0.0366 TKTL1 0.047 0.041 1.146 0.4543

Expression of PPP genes in paired HCC and NT liver tissues were analyzed by transcriptome sequencing. P values were calculated by paired t test.

that BACH1 binds strongly to two of the seven putative AREs TKT expression (Fig. 2E and Fig. S3). Coincidentally, the ARE that are located in intron 1 and intron 4 of TKT (Fig. 2B). BACH1 in intron 4 of TKT was also recently found to be a binding site for has been shown to inhibit an antioxidant gene, NAD(P)H NRF2 (13). dehydrogenase, quinone 1 (NQO1), through competitive binding with a transcriptional activator, Nuclear Factor, Erythroid 2-Like Knockdown of TKT Suppresses HCC Cell Proliferation by Inducing ROS 2 (NRF2) (12). Under normal conditions, NRF2 is ubiquitinated Accumulation and ROS-Associated Cell-Cycle Delay. Given that TKT by KEAP1, leading to proteosomal degradation of NRF2. Oxi- is located in a pivotal position connecting PPP and glycolysis, we dative stress inactivates KEAP1, thereby allowing NRF2 to enter postulated that TKT overexpression in cancer would substantially the nucleus and initiate transcription of genes involved in affect the metabolic machinery of cancer cells. To address this, we counteracting oxidative stress. We therefore questioned if TKT, established TKT stable knockdown cells in two different HCC like other antioxidant genes, was regulated by the KEAP1/NRF2 cell lines, MHCC97L and SMMC cells, by short-hairpin RNA pathway. By ChIP assay, we confirmed that NRF2 bound to the (shRNA) (Fig. 3 A and B). Two independent shRNA sequences two strong BACH1-binding sites in intron 1 and in intron 4 of (shTKT-1 and shTKT-2) profoundly suppressed HCC cell pro- TKT (Fig. 2C). Of note, BACH1 and NRF2 also bound to the liferation in vitro (Fig. 3C). 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol- other five putative AREs at a weaker affinity, as reflected by the 4-yl)Amino)-2-Deoxyglucose (2-NBDG) staining confirmed that fold of enrichment values (Fig. S2 B and C). To validate that knockdown of TKT reduced glucose uptake in HCC cells (Fig. BACH1 and NRF2 antagonistically regulate TKT expression 3D). We also found that knockdown of TKT suppressed NAPDH through competitive binding, we performed ChIP assay with the production and increased ROS production in HCC cells (Fig. 4 NRF2 antibody on BACH1 stable knockdown cells. Knockdown A–C). ROS accumulation caused by TKT knockdown could be of BACH1 enhanced binding of NRF2 to TKT (Fig. 2D). Fur- rescued by the antioxidant reduced glutathione (GSH) (Fig. 4D). thermore, stable knockdown of BACH1 and KEAP1 enhanced Next, we asked if the ROS accumulation caused by TKT knock- TKT expression, whereas stable knockdown of NRF2 suppressed down affects the cell-cycle profile of HCC cells. Nocodazole was TKT expression, suggesting that BACH1 and KEAP1 negatively used to synchronize all of the HCC subclones in the G2/M phase regulated TKT expression, whereas NRF2 positively regulated (Fig. S4). Cells were then released from nocodazole treatment for

Table 2. Clinicopathological correlation of TKT overexpression in human HCC Mean of −ΔΔCt, Clinicopathological features Status Number of cases T-NT P value

Gender Male 79 1.26 0.523 Female 22 0.98 Venous invasion Absent 43 0.61 0.002** Present 55 1.69 Tumor encapsulation Absent 64 1.51 0.025* Present 33 0.69 Tumor microsatellite formation Absent 48 0.77 0.013* Present 49 1.62 Hepatitis B surface antigen Absent 23 1.63 0.129 Present 71 1.00 Direct liver invasion Absent 54 0.97 0.130 Present 34 1.50 Cellular differentiation by I and II 46 1.08 0.529 Edmondson grading III and IV 51 1.30 Tumor size ≤5 cm 36 0.70 0.028* >5 cm 61 1.49 Cirrhosis Absent 46 1.35 0.483 Present 52 1.10 Tumor stage I and II 35 1.73 0.066 III and IV 63 1.77 CELL BIOLOGY

mRNA expression of TKT in paired HCC tumor and NT samples was measured by qRT-PCR. *P < 0.05, **P < 0.01, Student’s t test.

Xu et al. PNAS | Published online January 25, 2016 | E727 Downloaded by guest on October 2, 2021 Fig. 2. The NRF2/KEAP1/BACH1 pathway regulated TKT expression in HCC cells. (A) Positions and sequences of two NRF2 and BACH1 binding motifs in TKT. Positions refer to transcription start site (TSS). Exons are indicated as black blocks. (B and C) Re- cruitment of BACH1 (B) and NRF2 (C) to the two identified binding motifs in TKT.(D) Recruitment of NRF2 to the binding motifs in TKT in MHCC97L-NTC or -shBACH1 cells. ChIP assay was performed with antibodies against IgG, NRF2, or BACH1. Fold of enrichment was normalized to the according IgG controls. (E) Protein expression of TKT in MHCC97L- NTC, -shNRF2, -shKEAP1, and -shBACH1 subclones. Intensities of bands were analyzed by Image J and normalized to corresponding NTC. *P < 0.05, **P < 0.01, ***P < 0.001 versus the corresponding IgG, Student’s t test. Data are presented as means ± SD.

9 h. Propidium iodide (PI) staining showed that more cells stayed amount of glucose entering the oxidative and nonoxidative arm, in the G1 phase in TKT knockdown cells compared with nontarget respectively (Fig. 5D). As expected, glucose was forced to enter control (NTC) cells (Fig. 4E). The addition of two mechanistically the oxidative arm upon knockdown of TKT, which may explain different antioxidants, GSH and N-acetyl-cysteine (NAC), allowed the accumulation of Ru5P/R5P in the oxidative arm (Fig. 5D). the TKT knockdown cells to reenter the S phase, suggesting that Studies have shown that accumulation of Ru5P/R5P reduced the ROS accumulation caused by TKT knockdown resulted in cell-cycle activity of enzymes in the oxidative arm (14, 15). We therefore delay, thereby inhibiting HCC cell proliferation (Fig. 4E). As TKT examined the effect of Ru5P/R5P on the activity of G6PD, a key was more abundantly expressed in HCC compared with liver tissues, enzyme in the oxidative arm. We found that Ru5P and R5P we postulated that knockdown of TKT would have a greater impact together modestly reduced the activity of G6PD in HCC cells on HCC cell lines than on normal liver cell lines. We further (Fig. S6B). Consistently, we found that the activity of G6PD was established TKT knockdown stable cells in a normal liver cell line, also slightly reduced upon TKT knockdown (Fig. S6C). Despite MIHA (Fig. S5A). Knockdown of TKT did not affect the pro- the accumulation of Ru5P/R5P, levels of purine metabolites in- liferative output, ROS, and NADPH levels in MIHA cells (Fig. cluding adenosine, AMP, ADP, ATP, GDP, and GTP were con- S5 B–D). sistently reduced upon TKT knockdown (Fig. S6D). The pattern of our metabolomic study in TKT knockdown HCC cells is in TKT Decreases Glucose Flux into Glycolysis and Increases Glutathione agreement with the metabolomic study in NRF2 knockdown lung Synthesis. To comprehensively elucidate the impact of TKT on the carcinoma cells reported by others (13), further reinforcing the metabolic pathways in cancer cells, we performed metabolomic regulatory role of NRF2 on TKT. profiling using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) in MHCC97L-NTC and -shTKT stable Knockdown of TKT Suppresses HCC Growth in Vivo. To confirm that cells (Fig. S6A). As TKT recycles intermediates back into the the metabolic changes caused by knockdown of TKT would oxidative arm through several steps in glycolysis (Fig. 5A), we impact the cancer growth in vivo, we used two animal models: an found that those glycolytic intermediates including G6P, F6P, s.c. tumor model and an orthotopic tumor model. In the s.c. fructose-1,6-biphosphate (F1,6P), and 3PG accumulated in the tumor model, which allows easy monitoring of tumor growth, we TKT knockdown cells (Fig. 5B). Lactate, the final glycolytic injected SMMC-NTC, –shTKT-1, and –shTKT-2 cells into the product, was significantly decreased in TKT knockdown cells (Fig. flanks of nude mice and allowed the tumors to grow for 28 d. 5B). These data were in line with those of our glucose uptake assay Knockdown of TKT significantly suppressed s.c. tumor growth and suggested that glycolytic flux was decelerated in TKT (Fig. 6 A and B). In the orthotopic tumor model, which better mimics knockdown cells. Meanwhile, the PPP intermediates, Ru5P and the tumor microenvironment of cancer, we injected luciferase- R5P, and R5P derivative, phosphoribosyl pyrophosphate (PRPP), labeled MHCC97L-NTC, –shTKT-1, and –shTKT-2 cells into were elevated (Fig. 5C), suggesting that TKT knockdown cells the left lobes of nude mice. Mice were killed 42 d after implan- may fail to direct the intermediates back to the glycolysis, leading tation. Knockdown of TKT profoundly suppressed the growth of to the accumulation of nonoxidative arm intermediates. To con- the primary tumor in the livers of mice (Fig. 6C). Furthermore, firm the direction of metabolic flux upon TKT knockdown, we knockdown of TKT drastically reduced the growth of metastatic 13 performed a carbon tracing experiment with [1,2- C2]-glucose lesions in the lungs of mice as reflected by Xenogen imaging and detected the amount of labeled Ru5P/R5P by liquid chro- (Fig. 6D). Histological analysis with hematoxylin and eosin 13 matography–mass tandem spectrometry (LC-MS). [1- C1]-Ru5P/ (H&E)-stained HCC sections suggested that the HCC tumors 13 R5P ([M+1]) and [1,2- C2]-Ru5P/R5P ([M+2]) indicate the derived from MHCC97L-NTC cells had more irregular tumor

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Fig. 3. Knockdown of TKT reduced HCC cell proliferation and glucose uptake in vitro. (A) mRNA expression of TKT in MHCC97L and SMMC-NTC, –shTKT-1, and –shTKT-2 subclones. HPRT was used as the internal control. (B) Protein expression of TKT in MHCC97L and SMMC-NTC, –shTKT-1, and –shTKT-2 subclones. β-actin was used as the loading control. (C) Cell proliferation assay in MHCC97L and SMMC- NTC, –shTKT-1, and –shTKT-2 subclones. (D) Glucose uptake assay was performed in MHCC97L and SMMC-NTC, –shTKT-1, and –shTKT-2 subclones. (Left) Representative flow cytometry analysis showing intensities of 2-NBDG in the indicated HCC subclones. (Right) Histograms summarize the 2-NBDG intensities (glucose uptake) in different HCC subclones. Relative values were calculated based on the according NTC subclones. *P < 0.05, ***P < 0.001 versus NTC. A, C, and D, Student’s t test. Data are presented as means ± SD.

growth fronts and were more aggressive than those derived from sitization effect of the TKT inhibitor oxythiamine (OT), a thia- MHCC97L-shTKT cells (Fig. 6E and Table 3). Metastatic foci mine antagonist, for Sorafenib. Cell proliferation assay revealed in the mice implanted with MHCC97L-NTC cells were signifi- that OT drastically sensitized SMMC and MHCC97L cells to cantly larger (Fig. 6F). Sorafenib treatment in vitro (Fig. 7E and Fig. S7E) and increased ROS accumulation in the Sorafenib-treated HCC cells (Fig. 7F). Inhibition of TKT Sensitizes Cancer Cells to Sorafenib Treatment. We Of note, we found that Sorafenib alone could induce ROS in both demonstrated that TKT promoted cancer growth by attenuating HCC cell lines. This observation has also been reported by Coriat oxidative stress. Increasing evidence has suggested that reduction et al. in some other HCC cell lines, such as Hepa1,6 and HepG2 of oxidative stress protects cancer cells from chemotherapy and (16), suggesting that the effect of Sorafenib in ROS induction is radiation therapy. In light of this, we asked if inhibition of TKT common and partially contributed to its suppressive effect in the would sensitize cancer cells to Sorafenib treatment, the only FDA- growth of HCC. To confirm the mechanisms by which Sorafenib approved drug in HCC. Sorafenib partially reduced the cell pro- induces cell death, we performed Annexin V and PI staining to liferation in SMMC-NTC cells but completely blocked that in evaluate the percentage of apoptotic HCC cells. We found that SMMC-shTKT cells (Fig. 7A). The levels of ROS in the SMMC- Sorafenib induced apoptosis especially in the TKT knockdown NTC and -shTKT cells treated with vehicle control and Sorafenib HCC cells (Fig. S8A). A BrdU proliferation assay further con- inversely correlated with their proliferative output (Fig. 7B). We firmed that Sorafenib profoundly reduced cell growth, especially implanted SMMC-NTC and –shTKT-1 subclones s.c. into nude in the TKT knockdown HCC cells, which could be significantly mice for easy monitoring of the tumor growth. Consistent with our rescued by GSH (Fig. S8 B and C). These data suggested that the in vitro observation, Sorafenib partially repressed the growth of sensitization effect upon TKT knockdown or inhibition is caused tumors derived from SMMC-NTC cells but completely retarded by ROS. Interestingly, the sensitization effect only holds true in the growth of tumors derived from –shTKT-1 cells in vivo (Fig. 7 C HCC cells but not in normal liver cell lines, MIHA and LO2,

and D). Similar results were observed in s.c. tumors derived from suggesting a therapeutic window for combined treatment of OT CELL BIOLOGY MHCC97L subclones (Fig. S7 A–D). We next examined the sen- and Sorafenib in HCC patients (Fig. S9 A–C). Concentrations of

Xu et al. PNAS | Published online January 25, 2016 | E729 Downloaded by guest on October 2, 2021 Fig. 4. Knockdown of TKT increased the intracellular ROS level and induced oxidative stress-associated G1 phase arrest. (A and B) Flow cytometry was performed to analyze the ROS levels in MHCC97L and SMMC-NTC, –shTKT-1, and –shTKT-2 subclones. (Left) Representative flow cytometry analysis showing

intensities of ROS (CM-H2DCFDA) in the indicated subclones. (Right) Histograms summarize the ROS levels in different HCC subclones. Relative values were calculated based on the according NTC subclones. (C) Relative NADPH/NADP+ ratio in MHCC97L and SMMC-NTC and –shTKT-2 cells. The NADPH/ NADP+ ratio was normalized to the corresponding NTC subclones. (D, Left) Representative flow cytometry analysis showing intensities of ROS (CM-

H2DCFDA) in MHCC97L-NTC and –shTKT-2 cells with vehicle or 10 μM GSH. (Right) Histogram summarizes the ROS levels. Relative values were calculated based on the ROS level in the NTC. (E) Representa- tive flow cytometry analysis showing intensities of PI in MHCC97L-NTC and –shTKT-2 cells treated with vehicle, 10 μM GSH, or 2.5 mM NAC. Cells were synchronized with nocodazole and released from nocodazole for 9 h for cell-cycle analysis. *P < 0.05, **P < 0.01, ***P < 0.001 versus NTC or as indicated by brackets, Student’s t test. Data are presented as means ± SD.

OT used in in vitro and in vivo experiments were based on the resulted in a decrease of NADPH and an increase of ROS, further IC50 values and results of toxicity tests in other studies (17, 18). suggesting that oxidative stress homeostasis is an important de- The s.c. tumor model further revealed that a combination treat- termining factor in cancer growth. Of note, one intriguing question ment of OT and Sorafenib was most efficacious in suppressing that remains unanswered is why there was an increase of oxidative tumor growth compared with the single treatment of either drug flux in the TKT knockdown cells but the level of NADPH, a me- (Fig. 7 G and H). Apart from Sorafenib, we used another more tabolite from the oxidative arm, decreased. One speculation is that well-characterized ROS inducer, tert-Butyl hydroperoxide (tBHP). knockdown of TKT may transiently increase oxidative flux, but Consistently, we found that knockdown of both TKT and OT could gradual accumulation of R5P and Ru5P would eventually block the increase the ROS level of HCC cells together with tBHP (Fig. S10). oxidative arm of PPP. Our current study only showed that G6PD This finding suggested that OT mayalsobeusedincombination enzymatic activity was slightly reduced upon treatment of R5P and with other existing drugs to raise the ROS level in cancer cells in a Ru5P. A more direct experimental approach to confirm our hy- similar approach as Sorafenib. pothesis in the future is to use [1-2H]-glucose to trace NADPH production in the TKT knockdown cells (20). Discussion The PPP connects with glycolysis through G6PD and TKT, There has been a revival of interest in metabolic reprogramming in which directly metabolize several glycolytic metabolites. Our cancers. In the past decade, tremendous efforts have been made to metabolomic profiling provided further evidence to support the provide molecular explanations for the Warburg Effect, which was intimate link between the PPP and glycolysis. We noticed that reported 60 y ago (19). Compelling evidence has suggested that the end product of aerobic glycolysis, lactate, was reduced upon aerobic glycolysis is achieved to maximize the production of mac- knockdown of TKT despite the levels of glycolytic intermediates romolecules and antioxidants. In fact, aerobic glycolysis is accom- including F1,6P were significantly elevated. This observation can plished together with changes in other metabolic pathways. Our be explained by the change of pyruvate kinase M2 (PKM2) ac- study has provided evidence that a pivotal enzyme in the PPP, tivity, which is tightly controlled by F1,6P. PKM2 is one of the TKT, is indispensable to cancer development. The PPP produces four pyruvate kinase isoforms that convert phosphoenolpyruvate two major products, ribose and NADPH, through the nonoxidative (PEP) to pyruvate. PKM2, the cancer-specific isoform, has lower arm and oxidative arm, respectively. Surprisingly, we showed that enzymatic activity compared with other pyruvate kinase isoforms knockdown of TKT drastically reduced tumor growth regardless of (21). Low activity of pyruvate kinase reduces the entrance of the accumulation of ribose intermediates, suggesting that ribose pyruvate into the TCA cycle, thereby decelerating the metabolic was not a limiting factor in cancer growth. Although our current flux of the TCA cycle and allowing TCA cycle intermediates to study has not provided data to show whether ribose accumulation be synthesized into biomass, such as lipid (22). For its low en- in the TKT knockdown cells is a result of decreased consumption zymatic activity, PKM2 favors cancer growth. F1,6P allosterically or increased production, this remains an interesting question to be activates PKM2 through inducing the tetramer formation of addressed in the future. On the other hand, TKT knockdown PKM2 (21). Therefore, F1,6P accumulation upon TKT knockdown

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Fig. 5. Knockdown of TKT altered glucose metabolism and glutathione metabolism. (A) Metabolic intermediates and reactions in the glycolysis and PPP. (B and C) Quantification of metabolic intermediates in glycolysis (B)andthePPP(C). (D, Left) 13 1,2- C2-glucose is converted to M+1 Ru5P/R5P through the oxidative arm and M+2 Ru5P/R5p through the nonoxidative arm. (Right) Quantification of mass iso- topomer distribution of Ru5P/R5P in MHCC97L-NTC 13 and -shTKT cells cultured in 1,2- C2-glucose for 12 h. M+0 indicates unlabeled Ru5P/R5P. M+1 indicates one carbon-labeled Ru5P/R5P. M+2 indicates two carbon-labeled Ru5P/R5P. 3PG, 3-phosphoglyceric acid; E4P, erythrose 4-phosphate; F1,6P, fructose 1,6-bisphosphate; F6P, frutose-6-phosphate; G3P, glyceraldehyde 3-phosphate; G6P, glucose-6-phosphate; PRPP, phosphoribosyl pyrophosphate; R5P, ribose-5-phosphate; Ru5P, ribulose-5-phosphate; S7P, sedoheptulose-7-phosphate; X5P, xylulose-5-phosphate. *P < 0.05, **P < 0.01, ***P < 0.001 versus NTC, Student’s t test. Data are presented as means ± SD.

may result in the activation of PKM2, leading to the decrease of effect of OT with inhibitors against the glutathione and/or thi- aerobic glycolysis and a reduction of lactate accumulation. We have oredoxin antioxidant pathways represents an exciting trans- recently reported that PKM2 is the major isoform in HCC (23) lational research direction. Furthermore, knowledge on the and TKT might indirectly regulate PKM2 activity through F1,6P. combined effects of these inhibitors with existing chemotherapy Further knowledge on the interplay of TKT and PKM2 will help to and targeted therapies will be important for the development of devise combined therapies against the PPP and glycolysis for new therapeutic strategies for cancers, especially in HCC pa- cancer patients. tients with very limited treatment options. The role of ROS in cancer development has been the subject It is worth mentioning that patients with PPP deficiency, the of contentious debate. A wealth of studies have suggested that G6PD deficiency that is an X chromosome-linked genetic dis- ROS drives mutations and activates signaling pathways that ease in G6PD, have not been found to be protected against promote cell proliferation (24). Recently, more studies have cancers (33). G6PD directly produces NADPH, which is the only provided evidence showing that excessive production of ROS is source of GSH in red blood cells. G6PD deficiency increases detrimental to cancer cells by triggering apoptosis and senes- – oxidative stress in red blood cells, causing hemolysis, which cence (25 27). NRF2 is characterized as an oncogene for its destructs red blood cells and releases hemoglobin to plasma. ability to buffer ROS (28). Recently, NRF2 has been reported to G6PD deficiency has not been found to be protective against transcriptionally activate some PPP genes, such as TKT, G6PD, cancers, possibly because of the compensatory effects from other and TALDO (13). Interestingly, p73 could also increase the NADPH-producing enzymes (20, 34, 35). Although large-scale metabolic flux in the PPP through initiating the transcription of G6PD (29). Although our study has showed the roles of TKT in epidemiological studies on the protective roles of G6PD de- redox homeostasis, further studies on other possible transcrip- ficiency against cancers are awaited, the pathophysiology of tional and posttranscriptional regulation of TKT will yield im- G6PD deficiency suggested that systemic suppression of portant knowledge on how cancer cells overcome ROS. NADPH production may adversely cause side effects similar to Recent studies have highlighted the disadvantages of antioxi- the symptoms of G6PD deficiency. Careful examination of the dant intake in cancer patients. Antioxidants have been shown to therapeutic window of drugs that target antioxidant production confer cancer stem cells’ radio resistance (30) and promote an- in cancer treatment should be performed before clinical trials. In chorage-independent growth of cancer cells (31). Our study has illustrated an example that a pathway that synthesizes antioxidants, the PPP, is critical to tumor development. Our finding Table 3. Results of orthotopic liver implantation from echoes a recent study that elegantly demonstrated the important MHCC97L-Luc-NTC and -shTKT clones roles of glutamate cysteine ligase, an enzyme that synthesizes Number of glutathione, in cancer initiation (32). Intriguingly, Harris et al. Number of Number of tumors with demonstrated that cotreatment of inhibitors against the gluta- Experimental tumors with tumors with microsatellite thione and thioredoxin antioxidant pathways could efficiently groups invasive growth front venous invasion formation block tumor growth even when drugs were administered after tumor onset (32). Similarly, our study, using HCC as a cancer NTC 5/5 2/5 4/5 shTKT-1 2/5 0/5 1/5 model, showed that blocking the PPP by the TKT inhibitor OT CELL BIOLOGY could synergize Sorafenib to halt cancer growth. The combined shTKT-2 2/5 0/5 4/5

Xu et al. PNAS | Published online January 25, 2016 | E731 Downloaded by guest on October 2, 2021 Fig. 6. Knockdown of TKT suppressed tumor growth and lung metastasis in vivo. (A and B) SMMC-NTC and –shTKT-1 cells (A) or SMMC-NTC and –shTKT-2 cells (B) were s.c. injected into flanks of nude mice. Tumor volumes were monitored for 28 d. (Left)Representa- tive pictures of tumors formed in nude mice. (Scale bar, 1cm.)(Middle) Growth curves of s.c. xenografts. (Right) Quantification of tumor mass on day 28. (C) Luciferase-labeled MHCC97L-NTC, –shTKT-1, or –shTKT-2 cells were orthotopically injected into left hepatic lobes of nude mice and allowed to grow for 42 d. (Left) Representative pictures of orthotopic xenografts. (Scale bar, 1 cm.) Right: quantification of tumor volume. (D, Left) Bioluminescent images of lung tissues. (Right) Quantification of bioluminescent intensities of lung tissues. (E) Representative pictures of H&E staining of tumor xenografts. Arrows indicate irregular growth front in the NTC group. (Scale bars, 200 μm.) (F)Repre- sentative pictures of H&E staining of lung tissues. Arrows indicate tumor cells found in the lung tissue. (Scale bars, 200 μm.) *P < 0.05, **P < 0.01 versus NTC, Student’s t test (N ≥ 5). Data are presented as means ± SD.

conclusion, results from this study underscore the protumorigenic Transcriptome Sequencing. Transcriptome sequencing was performed in 16 roles of antioxidants in cancer development and support the cases of HCC tissues and paired NT liver tissues. TruSeq Standard mRNA notion that antioxidant intake may not be definitely beneficial to Sample Prep Kit (Illumina) was used to prepare the polyA + mRNA library. We cancer patients. performed 100-bp paired-end sequencing in Illumina HiSeq2000 by Axeq Technologies. Data were analyzed by the TopHat-Cufflinks pipeline (37), and Materials and Methods values were indicated by FPKM. Patients. Use of human clinical specimens in this study was approved by the TCGA and Oncomine Data. Transcriptome sequencing data of 50 cases of Institutional Review Board of the University of Hong Kong and Hospital paired HCC and NT tissues were retrieved from TCGA via the Broad Institute Authority of Hong Kong (HKU/HA HKW IRB; ref. no. UW 09–185). The pa- (gdac.broadinstitute.org/). mRNA expression levels of the PPP genes in dif- tients were explained, and signed consent forms to acknowledge, the use of their resected tissues for research purposes. ferent cancer types were retrieved from the Oncomine database (https:// www.oncomine.org//resource/login.html). Ethical Statement. Animal experiments were performed following UK Co- ordinating Committee on Cancer Research (CCCR) PMID: 9459138 Guidelines Clinicopathological Correlation Analysis. Clinicopathological features of HCC for the Welfare of Animals in Experimental Neoplasia (36) to ensure minimal patients including venous invasion, tumor encapsulation, tumor micro- suffering of animals. The experimental procedures of the animal studies satellite formation, hepatitis B surface antigen, direct liver invasion, cellular – – were approved by the Committee on the Use of Live Animals in Teaching differentiation, tumor size, and pathological tumor node metastasis (pTNM) and Research of the University of Hong Kong. tumor stage were graded and analyzed by a pathologist as previously described (38). Clinicopathological correlation was performed with HCC Patient Samples and HCC Cell Lines. Clinical HCC and corresponding ad- SPSS20.0 software. jacent NT liver tissue were obtained from patients at Queen Mary Hospital, Pamela Youde Nethersole Eastern Hospital, and Queen Elizabeth Hospital in ChIP Assay. Cells were cross-linked with formaldehyde and sonicated. Sheared Hong Kong. Tissue specimens were snap-frozen in liquid nitrogen and stored DNA was incubated with antibodies against NRF2 (Abcam), BACH1 (Santa at –80 °C for RNA or protein extraction. Human HCC cell lines SMMC and Cruz), or IgG (Santa Cruz). DNA–protein–antibody complexes were incubated MHCC97L were gifts from the Shanghai Institute of Cell Biology (Chinese with ChIP beads of protein A/G (Merck Millipore). Beads were washed with Academy of Sciences) and Fudan University of Shanghai, respectively. gradients of salt buffer and eluted in 1% SDS/NaHCO3. ChIP DNA was analyzed by qRT-PCR with primers amplifying the putative regions (Table S1). RNA Extraction, Reverse Transcription PCR, and Quantitative Real-Time PCR. RNA was extracted from clinical specimens or HCC cell lines by TRIzol re- Establishment of Stable Knockdown Clones. pLKO.1-puro vectors encom- agent (Life Technologies) and reverse-transcribed by the GeneAmp PCR passing shRNA targeting TKT, NRF2, KEAP1, BACH1 (Table S2), or a nontarget Reagent Kit (Applied Biosystems). SYBR Green qPCR Master Mix (Applied control (Sigma Aldrich) were stably transfected into HCC cell lines by a Biosystems) and corresponding primers (Table S1) were used for quantitative lentiviral-mediated approach, as we previously described (39). HCC cells that real-time PCR (qRT-PCR). stably expressed the vectors were selected by puromycin.

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Fig. 7. Genetic knockdown and pharmacological inhibition of TKT sensitized HCC cells to Sorafenib treatment via enhancing oxidative stress. (A) Pro- liferation curves of SMMC-NTC and –shTKT-2 cells in the presence of vehicle DMSO (Ctrl) or 6 μM Sorafenib (Sora). (B) Representative flow cytometry histogram (Left) and quantification (Right) of ROS staining in SMMC-NTC and –shTKT-2 cells treated with vehicle (Ctrl) or 6 μM Sora for 24 h. (C and D) Growth curves of s.c. xenografts derived from SMMC-NTC or –shTKT-1 cells in nude mice that were administered the control vehicle (Ctrl) or 30 mg/kg/d Sora for 14 d. (D, Left) Representative picture of tumor xenografts. (Right) Quantification of tumor mass (n = 6). (Scale bar, 1 cm.) (E) Proliferation curves of SMMC cells treated with vehicles, 6 μM Sora, 5 mM OT, or 6 μM Sora and 5 mM OT (Sora + OT). (F) Representative flow cytometry histograms of ROS staining in MHCC97L (Left) and SMMC (Right) cells treated with vehicles, 6 μM Sora, 5 mM OT, or 6 μM Sora and 5 mM OT (Sora + OT) for 24 h. (G) Growth curves of s.c. xenografts derived from parental SMMC cells in mice that were administered the vehicles (Ctrl), 30 mg/kg/d Sora, 80 mg/kg/d OT, or 30 mg/kg/d Sora and 80 mg/kg/d OT (Sora + OT) for 18 d. (H, Left) Representative pictures of tumor xenografts. (Right) Quantification of tumor mass (n = 12). (Scale bar, 1 cm.) *P < 0.05, **P < 0.01, ***P < 0.001 versus Ctrl. Student’s t test. Data are presented as means ± SD.

Cell Proliferation Assays. For cell counting assays, an equal number of cells was calculated by the following equation: (intensity of heated)/(intensity of seeded in a 12-well plate 24 h before the proliferation assay. The cell number unheated – heated samples). For glucose uptake assays, cells were stained in each well was counted daily by using the Z1 Particle Counter (Beckman with 100 μM 2-NBDG (Life Technologies) and analyzed by BD FACSCanto II Coulter) for 4–5 d. The BrdU assay (Roche) was performed based on the Analyzer. Enzymatic activity of G6PD was measured by the G6PD activity manufacturer’s instructions. Data were from three independent experi- colorimetric assay kit (Biovision) according to the manufacturer’s instruc- ments and are presented as means ± SD. tions. For R5P/Ru5P treatment, cell lysates were incubated with R5P (Sigma) and Ru5P (Sigma) for 30 min at room temperature before addition of the Protein Extraction and Immunoblotting. Protein was extracted from human substrate mix. HCC samples or HCC cells using radio-immunoprecipiation assay (RIPA) lysis buffer in the presence of PhosSTOP phosphatase inhibitor mixture (Roche) Cell-Cycle Analysis. Cell-cycle distribution was analyzed by PI (Calbiochem) and protease inhibitor mixture (Roche). Equal amounts of protein were staining. Cells were synchronized in the G2/M phase by 18 h of Nocodazole subjected to electrophoresis using 10% (vol/vol) SDS/PAGE. Antibodies (200 ng/mL) treatment. Synchronized cells were released from Nocodazole against TKT (Abcam) and β-actin (Sigma) were used for Western blotting. for 9 h and harvested. Cells were fixed with 75% (vol/vol) ethanol for at least 1 h and incubated with 50 μg/mL PI for 30 min. The PI signal was measured by ROS, NADPH, Glucose Uptake, and G6PD Enzymatic Assays. For ROS assays, an BD FACSCanto II Analyzer. equal number of cells were stained with 2 μM chloromethyl-2′,7′-dichlor-

odihydrofluorescein diacetate (CM-H2DCFDA) (Life Technologies) and ana- Apoptosis Analysis. Cells were seeded for 24 h and incubated with serum-free + lyzed by BD FACSCanto II Analyzer. NADPH/NADP ratios were measured by culture medium containing Sorafenib or an equal amount of DMSO. After the NADPH Colorimetric Assay Kit (Biovision) according to the manufac- 24 h, cells were trypsinized and suspended in Annexin V binding buffer (BD turer’s instructions. In brief, HCC cells were lysed by five repeated freeze/ Science) containing Annexin V-FITC (MBL International) and PI (BD Science) thaw cycles in extraction buffer. A portion of the samples were heated at for 15 min at room temperature based on the manufacturers’ instructions. 60 °C for 30 min for NADP decomposition. A portion of the samples remained Samples were immediately analyzed by the BD FACSCanto II Analyzer. unheated. Heated and unheated samples were incubated with NADP cycling buffer for 5 min and mixed with NADPH developer. NADPH and NADP in- Metabolomics. To extract intracellular metabolites, MHCC97L-NTC or MHCC97L- CELL BIOLOGY + tensities were measured using a plate reader at OD450. NADPH/NADP was shTKT cells were washed with 5% (wt/wt) mannitol and extracted with

Xu et al. PNAS | Published online January 25, 2016 | E733 Downloaded by guest on October 2, 2021 13 13 methanol in the presence of 10 μM of internal control solution (compound R5P, [1- C1]-R5P, [1,2- C2]-R5P, and compound A1 were set at 229/97, 230/ C1 with the m/z at 182.048 and compound A1 with the m/z at 231.070), 97, 231/97, and 231/80, respectively. The collision energies were also set at 16 which was provided by Human Metabolome Technologies. Extracted solu- eV and 35 eV for R5P and compound A1, respectively. tion was collected and centrifuged. Supernatants were filtered by a centri- fuge filter unit (Human Metabolome Technologies). Filtered extracts were Animal Experiments. For the s.c. tumor model, 1 × 106 HCC cells were sus- dried up by a centrifugal evaporator. Extracted metabolites were analyzed pended in 100 μL PBS and s.c. injected into BALB/cAnN-nu (nude) mice. Tu- by CE-TOFMS by Human Metabolome Technologies. mor volume was measured by electronic caliper. For the orthotopic tumor model, 1 × 106 luciferase-labeled MHCC97L cells were suspended in 100% × 6 Metabolic Flux Analysis. The 1.5 10 MHCC97L-NTC and -shTKT cells were Matrigel (BD Bioscience) and orthotopically injected into the left lobes of the 13 cultured in DMEM (Sigma, D5030) with 25 mM [1,2- C2]-glucose (Cambridge liver of nude mice. At 42 d after implantation, mice were injected with Isotope Laboratories) and 4 mM L-glutamine in 10% dialyzed FBS (HyClone) 100 mg/kg D-luciferin (PerkinElmer), and bioluminescence was measured by in 10-cm plates for 12 h. After cells were quenched with HPLC-grade Xenogen IVIS100. Tumors and lungs were harvested for ex vivo imaging and methanol and dehydrated completely, samples were sparked with 10 μLof histology. Tissues were fixed in 10% formalin and stained with H&E for compound A1 (2 ppm) as the internal standard. The samples were then histological analysis. briefly sonicated, vortexed, and centrifuged at 15,133 × g for 20 min. The supernatants were collected and dehydrated and dissolved in 200 μL of 80% methanol. The samples were vortexed and centrifuged at 15,133 × g for Drug Treatment. Sorafenib (LC laboratories) was dissolved in DMSO. OT 20 min, and the supernatants were collected for ultra performance liquid (Sigma), GSH (Sigma), and N-acetyl-L-cysteine (Sigma) were dissolved in H2O. chromatography (UPLC)-MS/MS analysis. Analysis was performed on a TSQ For in vitro experiments, Sorafenib, OT, GSH, N-acetyl-L-cysteine, and vehi- Quantiva triple quadrupole mass spectrometer (Thermo Fisher Scientific) via an cles were added to cells for proliferation assay, ROS assays, or cell-cycle electrospray interface, operating in negative ionization mode and configuring analysis. For in vivo experiments, nude mice were administered orally with in selective reaction monitoring mode. The metabolite separation was per- Sorafenib, and intraperitoneally with OT and vehicle (0.9% NaCl) based on formed using a SeQuant ZIC-HILIC column (3.5 μm, 100 × 2.1 mm) (Merck). The the therapeutic regimens shown in Fig. 7. mobile phases consisted of acetonitrile (A) and 20 mM ammonium acetate in water (B). The gradient elution program initiated from 90% (vol/vol) A and ACKNOWLEDGMENTS. We thank Dr. Sandy Leung-Kuen Au for her technical held for 1 min, decreased to 60% (vol/vol) A in 7 min, and held for 5 min, then support and advice in the animal experiments. We also thank the Faculty increased to 90% (vol/vol) for re-equilibrium for 5 min with a flow rate of Core Facility, University of Hong Kong Li Ka Shing Faculty of Medicine for its 0.2 mL/min. Mass spectrometric conditions were optimized for each me- support in the flow cytometry analysis and Xenogen imaging. This work was supported by the Health and Medical Research Fund (HMRF-02132696), SK tabolite by using the reference standard. Spray voltage, vaporizer tem- Yee Medical Research Fund 2011, University Development Fund of the perature, sheath gas, auxiliary gas, and capillary temperature were set at University of Hong Kong, Lee Shiu Family Foundation, National Natural – 2,300 V, 350 °C, 35 arb, 10 arb, and 320 °C, respectively. The LC-MS/MS Science Foundation of China (NSFC-21377106), and Hong Kong Research data were acquired and processed with LCquanTM software version 2.5.6 Grant Council Collaborative Research Fund (HKBU5/CRF/10). I.O.-L.N. is Loke (Thermo Fisher Scientific). The selective reaction monitoring transitions of Yew Professor in Pathology.

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