APC/C-CDH1–Regulated Idh3b Coordinates with the Cell Cycle To

Published OnlineFirst May 3, 2019; DOI: 10.1158/0008-5472.CAN-18-2341

Cancer Molecular Cell Biology Research

APC/C-CDH1–Regulated IDH3b Coordinates with the Cell Cycle to Promote Cell Proliferation Qingnan Wu1, Weimin Zhang2, Liyan Xue3, Yan Wang2, Ming Fu1, Liying Ma1, Yongmei Song1, and Qi-Min Zhan1,2

Abstract

Metabolic activities are often accompanied by cell-cycle progression, yet known connections G M Early G Late G S between these two processes remain limited. Here, 2 1 1 we identiﬁed the isocitrate dehydrogenase 3b (IDH3b) as a novel substrate of anaphase-promot- α-KG CDH ing complex/cyclosome (APC/C)-CDH1 and an APCIC APCIC 1 CDH1 important regulator of the cell cycle. In esophageal IDH3β squamous cell carcinoma (ESCC), IDH3b was IDH3β α-KG posttranslationally upregulated in late G1 phase, – Succinate and overexpression of IDH3b accelerated G1 S β Isocitrate IDH3 Ub Ub Ub transition, contributing to the promotion of cell TCA in vitro in vivo proliferation and . a-Ketoglutarate Cycle Fumarate (a-KG), a crucial metabolite in tricarboxylic acid Citrate (TCA) cycle, was dependent on IDH3b level and Degradation Malate partially accounted for IDH3b-mediated cell Oxaloacetate growth. IDH3b expression increased PFKFB3 pro- β tein levels and enhanced glucose uptake, and high The APC/C-CDH1 substrate IDH3 accumulates in late G1 and accelerates G /S transition by raising intracellular levels of α-KG. expression of IDH3b correlated with poor survival 1 in patients with ESCC, suggesting a potential appli- © 2019 American Association for Cancer Research cation of IDH3b in prognosis. Overall, our results highlight a new molecular connection between cell-cycle regulation and the TCA cycle in ESCC.

Signiﬁcance: These ﬁndings show that IDH3b is an APC/C-CDH1 substrate and is expressed in a cell-cycle–dependent manner, highlighting novel molecular cross-talk between the TCA cycle and cell cycle in cancer cells. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3281/F1.large.jpg.

Introduction

1State Key Laboratory of Molecular Oncology, National Cancer center/National Cancer cells require core metabolic functions to produce ener- Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of gy, control redox, and assimilate biomass for supporting excessive Medical Sciences and Peking Union Medical College, Beijing, China. 2Key Lab- cell growth (1). It is outlined that cell-cycle modulators participate oratory of Carcinogenesis and Translational Research (Ministry of Education/ in coordinating metabolism to promote cancer initiation and Beijing), Laboratory of Molecular Oncology, Peking University Cancer Hospital & progression (2). In recent studies, cyclins and cyclin-dependent Institute, Beijing, China. 3Department of Pathology, National Cancer center/ National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy kinases (CDKs) were frequently involved in various metabolic of Medical Sciences and Peking Union Medical College, Beijing, China. processes, such as glycometabolism (3, 4), lipogenesis (5), amino acid metabolism (6), and mitochondrial activity (7). Conversely, Note: Supplementary data for this article are available at Cancer Research ﬂ Online (http://cancerres.aacrjournals.org/). metabolic alterations also in uence cell cycle during tumorigenesis (8). Pyruvate kinase muscle isoform M2, a well-known Q. Wu and W. Zhang contributed equally to this article. metabolic enzyme, was demonstrated to regulate not only Corresponding Author: Qi-Min Zhan, State Key Laboratory of Carcinogenesis G1–S transition but mitosis progression (9, 10). Nevertheless, and Translational Research (Ministry of Education/Beijing), Laboratory of the coordinated temporal regulation between cell cycle and Molecular Oncology, Peking University Cancer Hospital & Institute, Beijing metabolism needs further explorations. 10021, China. Phone: 8610-6776-2694; Fax: 8610-6771-5058; E-mail: [email protected] With two essential coactivators, CDC20 and CDH1 (11), the E3 ubiquitin ligase anaphase-promoting complex (APC/C or cyclo- Cancer Res 2019;79:3281–93 some) targets crucial cell-cycle proteins for proteasomal degrada- doi: 10.1158/0008-5472.CAN-18-2341 tion to ensure accurate timing of mitotic events temporally and 2019 American Association for Cancer Research. spatially (12). Besides canonical functions of APC/C in

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controlling cell-cycle transition, APC/C carries out additional motifs by R algorithm: those genes containing both destruction function in linking cell cycle and metabolic network in human box (D box, RXXLXXXXN/D/E, where X indicates any amino acid) cancers (13). APC/C-CDH1 takes 6-phosphofructo-2-kinase/fruc- and the Lys-Glu-Asn box (KEN box, KENXXXN) were enrolled in tose-2, 6-bisphosphatase, and isoform 3 (PFKFB3) as a substrate Set 1. Next, according to our previous study (27), we extracted the to impact glycolysis and pentose phosphate pathway (14), and it copy number variation (copy number gain and loss) information also targets glutaminolytic enzyme glutaminase 1 (GLS1) to affect of the 2,752 metabolism genes, and genes with variation frequen- glutaminolysis (15). Despite those APC/C-influenced metabolic cy larger than 20% among all samples were placed into Set 2. pathways, the molecular connection between cell cycle and cell Genes in the intersection of Set 1 and Set 2 were put forward for metabolism is still far from understood, especially whether the further study. tricarboxylic acid (TCA) cycle has undergone direct regulation by APC/C is unclear. As a key metabolic pathway, TCA cycle is a series Cell culture and treatment of reduction and oxidation reactions that produce energy and ESCC cell lines such as KYSE30, KYSE410, and KYSE510 were intermediates used for biosynthetic processes (16). Early opinions provided by Y. Shimada of Kyoto University (Kyoto, Japan). They agreed that cancer cells preferentially utilized aerobic glycolysis were tested for Mycoplasma contamination using Mycoplasma and bypassed the TCA cycle, but recent opinions claimed that Real-time PCR Detection Kit (Applied Biosystems) and were certain cancer cells were dependent heavily on the TCA cycle to authenticated using short tandem repeat profiling. Cells were synthesize precursors for cell proliferation (17, 18). Therefore, it is cultured in RPMI1640 (Gibco) with 10% FBS. The stable knock- noteworthy to pour attention on cell-cycle–regulated TCA cycle down cell line KYSE510 was cultured in RPMI1640 (Gibco) with enzymes. 10% FBS and 0.5 mg/mL puromycin. All of these cells were The isocitrate dehydrogenase 3b (IDH3b) is a subunit of the maintained at 37 C with 5% CO2. For G2–M phase arrest, cells IDH3 heterotetramer composed of two a, one b, and one g were treated with 200 ng/mL nocodazole (Sigma) for 18 hours. subunits. The IDH family catalyzes oxidative decarboxylation of For double thymidine treatment, cells were treated with 2 mmol/L isocitrate to a-ketoglutarate (a-KG). The two members, IDH1 and thymidine (Sigma) for 18 hours, fresh medium for 8 hours, and IDH2, were distinguished for their mutations in multiple types of then 2 mmol/L thymidine for another 24 hours. Other reagents cancers (19, 20), highlighting that dysfunction of metabolic used were as follows: cycloheximide (100 mg/mL, Sigma), MG132 enzymes leads to cancer formation. Although IDH1 and IDH2 (10 mmol/L, Sigma,), a-KG (2 mmol/L, Sigma), and succinyl are commonly discussed and even IDH3a are reported (21, 22), phosphonate (0.1 mmol/L, MCE). the implication of IDH3b in tumorigenesis still remains obscure. Esophageal cancer is a common gastrointestinal cancer and the Plasmid construction and infection sixth leading cause of cancer mortality (23). Half of global The plasmid IDH3A-flag, IDH3B-flag, and IDH3G-flag were esophageal cancer cases happen in China and esophageal squa- purchased from GeneChem Co. Ltd., which contained the full- mous cell carcinoma (ESCC) is the major subtype histologically. length coding region of IDH3A, IDH3B, IDH3G and fused with Accounting for the high frequency of metastasis and relapse, ESCC triple flag tags in the C-terminal, respectively. Lentiviral over- has poor prognosis even after potentially curative therapies. To expression plasmid pLVX-IDH3b was constructed by inserting the have a better understanding of ESCC progression, a number of corresponding Human cDNAs into the EcoRI/BamHI sites of whole-genome analysis of ESCC have been undertaken to date in pLVX-puro vector. Short hairpin RNA (shRNA) oligonucleotide China, which would provide effective approaches to improve was designed to target the 30UTR region of IDH3B (shIDH3b:50- diagnosis and therapy (24, 25). However, few researches were CGGCATCTTAATCTTGAGTAT-30, shIDH3b-2: 50-GCAGAATCC- conducted to clarify the metabolism reprogramming during TTACCAGTTT-30), which was constructed in pLKO.1 puro vector. malignant transformation in ESCC. The pLVX-IDH3B and pLKO.1-shIDH3B plasmids were then To further characterize the intersection between cell-cycle packaged into lentivirus system with pMD2.G and pSPAX2 machinery and TCA cycle metabolism in ESCC, we found that vectors using HEK293T cells, respectively. The overexpression IDH3b contained canonical APC/C recognition motif and made or RNAi lentiviruses were transfected into cells using polybrene the first demonstration that IDH3b was a novel APC/C substrate. (8 mg/mL) to enhance infection efficiency. Puromycin (2 mg/mL) We showed that IDH3b was posttranslationally upregulated was added to the cells for 72 hours to screen for stably infected in late G1 phase and promoted cell proliferation by accelerating cells. The CDH1-myc and CDC20-myc plasmids were con- G1–S transition. In addition, a-KG level was positively dependent structed by our laboratory previously (28), and the target on IDH3b and greatly contributed to the IDH3b-mediated cell sequences of siCDH1 are listed as follows: si1: 50-GCGTGA proliferation. We also found that IDH3b could influence PFKFB3 ACTTCCACAGGAT-30;si2:50-GCTCCCAAGTGTGCAATCT-30. and enhance glucose uptake. In addition, altered expression of IDH3b was associated with poor prognosis of ESCC clinically. Immunoblotting and immunoprecipitation Collectively, our results rendered a new molecular connection For immunoblotting, cell lysates were prepared using PBS with between core cell-cycle machinery and TCA cycle regulation. 1% NP-40, protease inhibitors cocktail (Roche). The lysates were resolved by SDS-PAGE and transferred to ployvinylidene difluor- ide membrane. Membrane were incubated with indicated primary Materials and Methods antibodies after blocking, and then incubated with horseradish Identified metabolism relative potential APC/C substrates peroxidase –conjugated secondary antibody. Luminescent Image Protein sequences of a previously curated list of 2,752 meta- Analyzer LAS-4000 (Fujifilm) was used to detect the chemilumi- bolic enzymes and transporters (26) were downloaded through nescence signal. the NCBI Batch Entrez tool (https://www.ncbi.nlm.nih.gov/sites/ For immunoprecipitation (IP), the cells were treated with IP batchentrez) and supplied for searching APC/C recognition buffer (20 mmol/L Tris/HCL, pH 7.6, 100 mmol/L NaCl, 20

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mmol/L KCl, and 1.5 mmol/L MgCl2, 0.5% NP-40) adding tion motifs, destruction box (D box, RXXLXXXXN/D/E, where X protease inhibitor cocktail. The whole-cell lysates were incubated indicates any amino acid), and the Lys-Glu-Asn box (KEN box; with protein A/G-Sepharose beads (Roche), pretreated with anti- ref. 29). A total of 116 proteins containing both KEN box and D IDH3b (Santa Cruz Biotechnology sc-55674 for IP, Abcam box were selected for further analysis (Set 1, Supplementary Table ab112544 for Western blot and IHC analyses), anti-CDC27 S1; (ii) copy number variation of these 2,752 genes were analyzed (Abcam ab10538), anti-APC2 (Santa Cruz Biotechnology sc- in our previous pooled ESCC sequencing cohort (27), with a 20984), anti-CDH1 (Sigma C7855), anti-CDC20 (Cell Signaling variation frequency cutoff (larger than 20%) yielding 440 candi- Technology 14866S), anti-ubiquitin (Santa Cruz Biotechnology date genes (Set 2, Supplementary Table S2); (iii) genes over- sc-9133), anti-Flag (Sigma F1804), or anti-Myc (Cell Signaling lapping of Set 1 and Set 2 were put forward for further study. Technology 2278S) antibody at 4C overnight. The beads were Accordingly, we figured out 17 cell-cycle–related metabolic genes, washed with cell lysis buffer and the immunoprecipitated sam- including 10 CNV gain genes (ATP13A3, PIK3R4, PLCH1, ples were analyzed by Western blot. TXNRD3, IDS, HS6ST2, RDH11, PLCG1, SLC12A5, and IDH3B) and 7 CNV loss genes (KCND1, AGPAT5, MOCOS, GALNT1, Ethics statement PDSS1, ATP10D, and LNPEP), suggesting their critical roles during Human ESCC samples were collected directly after surgical ESCC progression (Fig. 1A). resection at Cancer Institute and Hospital, Chinese Academic As previous work reported that different TCA cycle metabolites of Medical Sciences & Peking Union Medical College (Beijing, peak in distinct cell-cycle phases (acetyl-CoA and citrate peak in China). Written informed consent was obtained from all patients G1–S, while malic acid and a-KG peak in late S, suggesting that the prior to the study. The use of the clinical specimens for research TCA cycle flux is regulated as cells progress through the cell cycle; purposes was approved by the Institutional Research Ethics ref. 30), accordingly, we treated cells with nocodazole to induce Committee. cell-cycle arrest. Cells were collected at indicated time after nocodazole release to analyze cell-cycle distribution, and cells at the Xenograft studies same timepoint were applied for TCA metabolites' detection, Six-week-old female BALB/c nude mice were used for tumor including citrate, a-KG, fumarate, and malate by commercial kits. formation. A total of 1 106 IDH3b stable overexpression cells The results showed that changing trends of a-KG, fumarate, and and control cells were injected subcutaneously into the right and malate were significantly correlated with S phase (Supplementary left dorsal flank, respectively. After 1 month, the mice were Fig. S1A–S1C). On the basis of these findings and our screened sacrificed to measure the tumor weight and tumor volume cal- genes, we focused on IDH3b, which encoded one of the rate- culated by the formula: p4/3 larger diameter smaller diam- limiting enzymes in TCA cycle, to explore the molecular linkage eter2. All animal care and procedures were in accordance with between TCA cycle and cell cycle. national and institutional policies for animal health and well- being. Mouse experimentations were approved by Cancer Insti- APC/C-CDH1 modulates IDH3b protein levels in late G1–S tute and Hospital, Chinese Academy of Medical Sciences and To ensure IDH3b is a bona fide APC/C substrate as expected, we Peking Union Medical College (Beijing, China). Three indepen- conducted IP assay and found endogenous IDH3b interacted with dent experiments were performed. two important components of APC/C (31, 32), APC2 and CDC27 (Fig. 1B and C). Next, we checked the oscillation of IDH3b protein Statistical analysis level during cell-cycle progression. KYSE30 cells were synchro- Statistical analysis was assessed by IBM SPSS Statistics 20 and nized in mitosis with nocodazole treatment, and endogenous GraphPad Prism 7. Two-tailed Student t test was used to analyze IDH3b protein level at indicated time after releasing the cells into the statistical significance of differences between experimental fresh medium was measured. Cell-cycle distribution was analyzed groups. The two-tailed Pearson x2 test was used to analyze the by flow cytometry using propidium iodide staining. The flow association of IDH3b expression and clinicopathologic para- cytometric analysis clearly showed G2–M phase arrest and IDH3b meters. The survival curves were plotted by using Kaplan–Meier appeared to be fluctuated in a cell-cycle–dependent way, peaking analysis and compared by log-rank test. Survival data were also in late G1–S and decreasing in mitosis (Fig. 1D). Moreover, in cells evaluated by multivariate Cox regression analysis. Each experi- released from G1–S block after double thymidine treatment, the ment was performed at least three times. Data are presented as similar oscillation of IDH3b was observed that its protein abun- mean SEM. Differences were considered significant when the P dance was highest in late G1 and early S-phase (Fig. 1E; Supple- < 0.05. mentary Fig. S2A). A detailed description of methods is described in Supplemen- It is reported that APC/C-CDC20 primarily works in early tary Materials and Methods. mitosis, while APC/C-CDH1 actives at the end of mitotic exit and early G1 phase (33). To figure out whether CDH1 or CDC20 plays a dominating role in regulating IDH3b, we showed that Results IDH3b interacted with CDH1, but not with CDC20 (Fig. 1F; Identified IDH3b as an APC/C candidate substrate Supplementary Fig. S2B). We performed proximity ligation assay Given the APC/C plays crucial roles in cell-cycle regulation, we (PLA) to show the interaction of IDH3b and CDH1 in situ sought to specifically determine the metabolic enzymes and (Fig. 1G). In addition, IDH3b protein abundance had no signif- transporters, which were regulated by APC/C and dysregulated icant changes in CDC20-overexpressed cells (Supplementary Fig. in ESCC. For this purpose, we set up numerous rigorous criteria to S2C), while it was reduced in CDH1-overexpressed cells (Fig. 1H). screen out the potential candidates: (i) protein sequences of a The mRNA levels of IDH3b remained unchanged (Supplementary previously curated list of 2,752 metabolic enzymes and transpor- Fig. S2D), suggesting a posttranscriptional regulation by APC/C- ters (26) were provided for searching canonical APC/C recogni- CDH1. Next, to explore when IDH3b interacted with CDH1 in the

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Figure 1.

APC/C-CDH1 modulates IDH3b protein levels in late G1–S. A, Overlapping genes of APC/C substrate candidates and genes with copy number alteration. Red, ampliﬁed genes; green, genes with CNV deletion. B, KYSE30 cell lysates were incubated with Protein A/G Sepharose conjugated with anti-IDH3b and anti-APC2. The immunoprecipitates were detected by immunoblotting. C, KYSE30 cell lysates were incubated with Protein A/G Sepharose conjugated with anti-CDC27 and anti-IDH3b antibody. The immunoprecipitates were detected by immunoblotting. D, KYSE30 cells were arrested in mitosis and released into fresh medium. Top, cells were collected for immunoblotting. Bottom, cells were stained with propidium iodide and their DNA content was determined by ﬂow cytometry. E, KYSE30

cells were arrested in G1–S boundary and released into fresh medium. Cells were collected every 2 hours for immunoblotting. F, KYSE30 cell lysates were incubated with Protein A/G Sepharose conjugated with anti-CDH1 and anti-IDH3b antibody. The immunoprecipitates were detected by immunoblotting. G, PLA signals of IDH3b and CDH1 in KYSE30 cells. H, KYSE30 cells were transfected with CDH1-myc plasmid, and IDH3b protein level was detected by immunoblotting. I, KYSE30 cells were transfected with siCDH1, and IDH3b protein level was detected by immunoblotting. J, IDH3b protein level in G1 phase was examined at indicated time after nocodazole (Noc) release in CDH1 knockdown cells. IP, immunoprecipitation; IB, immunoblotting; Thy, thymidine.

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cell cycle, we synchronized cells in G1–S with double thymidine depleted cells showed delayed accumulation of cyclin A as well as treatment and released into fresh medium: cells were collected to Geminin and delayed degradation of cyclin D, which further perform PLA at 0, 2, and 5 hours after thymidine release. The PLA confirmed the role of IDH3b in G1 phase promotion (Supple- signals were strongest at 0 hour and weakened gradually at 2 and 5 mentary Fig. S4C). hours (Supplementary Fig. S3A). Besides, we also performed PLA On account of promoting G1–S transition and raising the cell in cells collected at 0, 2, and 5 hours after nocodazole treatment proportion in mitosis, we speculated that IDH3b could facilitate release. The PLA signals were weakest at 0 hour and enhanced cell proliferation. Thus, we evaluated the ability of IDH3b on the gradually at 2 and 5 hours (Supplementary Fig. S3B). Therefore, proliferation and clonogenicity in ESCC cells. Notably, IDH3b- our results suggested that IDH3b:CDH1 interacted in G1 phase, in flag–overexpressed cells displayed more colonies than control which APC/C-CDH1 was most active. Consistently, CDH1 knock- group, while cells that IDH3b knocked down represented fewer down increased IDH3b level as expected (Fig. 1I). We also treated colonies (Fig. 3C; Supplementary Fig. S4D). In the cell growth CDH1 knockdown cells with nocodazole and released. IDH3b curve detected by xCELLigence System, it showed an identical level was increased in G1 phase and hardly changed in cells in consequence that IDH3b improved cell proliferation (Fig. 3D; which CDH1 was knocked down, indicating that the APC/C- Supplementary Fig. S4E) and the nondegradable mutant IDH3b CDH1 is the major regulator of IDH3b in G1 phase (Fig. 1J). had a more striking effect on increasing cell proliferation (Sup- As the APC/C is an E3 ubiquitin ligase, we next investigated plementary Fig. S4F). To strengthen our conclusion, xenograft whether the degradation of IDH3b was in a proteasomal- study in vivo was employed. The stably overexpressing IDH3b dependent pathway. We measured the half-life of IDH3b by KYSE30 and KYSE410 cells were subcutaneously injected in nude inhibiting protein synthesis with cycloheximide. However, pre- mice and the volumes of xenograft tumors were measured after vious work revealed that proteins involved in central metabolism, one month, and tumors in IDH3b-overexpressed group were including glycolysis and TCA cycle, were extraordinarily stable distinctly bigger than the control group (Fig. 3E; Supplementary and had long half-life time (34). Thus, we conducted long-time Fig. S5A). Taken together, we conclude that IDH3b promotes incubation and saw more stable half-life time when combined G1–S transition and promotes cell proliferation. with proteasome inhibitor MG132 (Fig. 2A). The results suggest that IDH3b decrease is due to proteasomal degradation, but not IDH3b-dependent a-KG generation accounts for improving transcriptional regulation. Moreover, we performed ubiquitin IP cell proliferation in the presence of CDH1 or CDH1 with MG132. IP of endogenous IDH3b converts isocitrate to a-KG in TCA cycle. Hence, we IDH3b was used to evaluate its ubiquitinated level. The ubiqui- examined the intracellular a-KG level in IDH3b-overexpressed tylation level of IDH3b was substantially increased when ectop- cells and control cells. It was clear that a-KG level was higher in the ically expressed myc-tagged CDH1 was combined with MG132 cells overexpressing IDH3b than that seen in the control cells (Fig. 2B). Moreover, clear interaction between ubiquitin and (Supplementary Fig. S6A). Conversely, the level of a-KG was IDH3b was exhibited (Fig. 2C). reduced when IDH3b was knocked down (Supplementary Fig. As IDH3b contains a potential APC/C-binding KEN-box and a S6B). In addition, we detected the levels of fumarate and malate, D-box degron motifs, we constructed mutations in IDH3b-flag two downstream metabolites in TCA cycle, under IDH3b over- plasmid in which both KEN-box and D-box were mutated expression. The fumarate and malate level were also mildly (Fig. 2D). After cycloheximide treatment, the ectopically increased after upregulating IDH3b (Supplementary Fig. S6C). expressed MUT-flag plasmid presented longer half-life time and Furthermore, IDH3a and IDH3g overexpression did increase the its protein level was more stable than IDH3b-flag when coex- amount of S-phase cells, and a-KG, fumarate, and malate levels pressed with CDH1-myc (Fig. 2E and F). Consistent with these were significantly increased in IDH3a- and IDH3g-overexpressing results, we also explored less ubiquitylation in MUT-flag with cells compared with the control cells (Supplementary Fig. S6D– MG132 (Fig. 2G). Collectively, our results uncover that IDH3b S6G). These results demonstrated that IDH3b, as a component of was a novel substrate of APC/C-CDH1 and degraded through IDH3, would contribute to oxidative metabolism in TCA cycle. APC/C-CDH1–mediated ubiquitin–proteasomal manner. Because a-KG is a crucial metabolite for cell proliferation and the a-KG level was upregulated by IDH3b, we hypothesized that IDH3b regulates G1–S transition and promotes cell the role of IDH3b in promoting cell proliferation might be proliferation mediated through a-KG generation. To probe this, we treated To explore the biological function of IDH3b in tumor progres- two cell lines with cell-permeable a-KG. We observed that a-KG sion, both IDH3b-flag plasmid and two IDH3b-targeted shRNA enhanced cell growth (Fig. 4A; Supplementary Fig. S7A), and the vectors were ectopically coexpressed in two ESCC cell lines (Sup- cell proportion in S-phase increased gradually along with the plementary Fig. S4A). First, we used nocodazole to cause cell arrest elevated a-KG concentrations in flow cytometric analysis (Fig. 4B; in G2–M phase and then refed with fresh medium: we observed an Supplementary Fig. S7B) and Edu incorporation assay (Fig. 4C; increased cell distribution in S-phase in IDH3b-flag–expressed Supplementary Fig. S7C). Consistently, the ability of colony cells compared with control cells (Fig. 3A). EdU (5-Ethynyl-20- formation in cells was enhanced after a-KG treatment (Fig. 4D; deoxyuridine) incorporation into cells released from G2–M phase Supplementary Fig. S7D). Therefore, a-KG was suggested to arrest was increased by IDH3b-flag (Fig. 3B) and decreased by modulate G1–S transition and thus augment cell proliferation in shIDH3b (Supplementary Fig. S4B), suggesting a role of IDH3b a dosage-dependent manner. To further validate a-KG produced in regulating S-phase entry. To further confirm the effect of by IDH3b was indeed involved in cell proliferation, we selected IDH3b knockdown on G1–S transition, we next generated a the most sufficient shRNA and supplied the IDH3b knockdown stably IDH3b-depleted KYSE510 cell line and treated it with cells with 2 mmol/L a-KG. The shRNA-mediated depletion of nocodazole, and cells were collected at 4, 6, 8, 12, and 14 hours IDH3b resulted in decreased cell growth as proven; however, after nocodazole release. The results displayed that the IDH3b- a-KG supplement partially rescued these diminished phenotypes

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Figure 2. IDH3b degrades in APC/C-CDH1–mediated ubiquitin–proteasomal manner. A, KYSE30 cells were treated with cycloheximide (CHX) or cycloheximide combined with MG132 and collected at the indicated time. IDH3b level was detected by immunoblotting. B, KYSE30 cells were transfected with CDH1-myc for 48 hours and incubated with 10 mmol/L MG132 for 6 hours prior to harvesting. Endogenous IDH3b was precipitated from the whole-cell extracts. C, Immunoprecipitation for endogenous IDH3b and ubiquitin. D, Mutations in the two degradation signals of IDH3b. The numbers on the top of the scheme denote the amino acid number at which the consensus sequence starts in relation to the start codon. E, IDH3b-flag and MUT-flag plasmids were transfected into KYSE30 cells and treated with cycloheximide. Cells were collected at the indicated time. The wild-type IDH3b and the mutated IDH3b level were detected by anti-Flag antibody. F, CDH1-myc coexpressed with IDH3b-flag or MUT-flag for 48 hours. Anti-Flag antibody was used to evaluate the wild-type IDH3b and the mutated IDH3b level. G, IDH3b-flag and MUT-flag plasmids were transfected into KYSE30 cells for 48 hours and treated with 10 mmol/L MG132 for 8 hours before harvest. Exogenous wild-type IDH3b and the mutated IDH3b were precipitated from the whole-cell extracts. Quantification was made by ImageJ. IP, immunoprecipitation; IB, immunoblotting.

of cell proliferation (Fig. 4E; Supplementary Fig. S7E) and cell- whereas it was decreased upon IDH3b interference (Fig. 5A cycle distribution (Fig. 4F and G; Supplementary Fig. S7F and and B). Thus, we sought to determine the change of enzymes S7G). Moreover, we found that the effect of IDH3b on prolifer- involved in glycolysis that provides material to TCA cycle. It is ation was attenuated by abolishing a-KG oxidation with a-KG shown that the expression levels of PFKFB3, which facilitates dehydrogenase inhibitor succinyl phosphonate (Supplementary glycolysis, were upregulated in IDH3b-flag–transfected cells. Fig. S7H; 35). Taken all together, our findings uncovered that Moreover, knockdown of IDH3b reduced PFKFB3 protein level IDH3b overexpression influenced S-phase entry, and the promo- (Fig. 5C). These results showed that PFKFB3 was positively tion on cell growth of IDH3b partially depended on a-KG correlated with IDH3b expression, accounting for the heightened production. glucose uptake to a certain degree. Because previous studies established that the nuclear PFKFB3 stimulated cell growth, we IDH3b enhances nuclear and cytoplasmic PFKFB3 level turned to measure the nuclear PFKFB3 protein level after intro- Given the regulation of IDH3b in cell cycle, we were curious ducing IDH3b-flag. We conducted immunofluorescence and about its role in metabolic pathways. Interestingly, we found the nuclear and cytoplasmic protein extraction assay after overex- glucose uptake was increased in IDH3b-overexpressed cells, pressed IDH3b-flag. Intriguingly, we found that high-level IDH3b

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Figure 3.

IDH3b regulates G1–S transition and promotes cell proliferation. A, Cells transfected with IDH3b-flag plasmid or control vector were synchronized in G2–M phase and then released into fresh medium. Cells were stained with propidium iodide and analyzed by flow cytometry. B, Cells transfected with IDH3b-flag plasmid and control vector were synchronized in G2–M phase and then released into fresh medium. After 20 hours, 50 mmol/L Edu incorporation was added into cell during the last 1.5 hours for culture. At least 300 cells were examined per slide. C, Colony-forming assays of cells transfected with IDH3b-flag plasmid and control. Representative images (left) and quantitative analysis (right) of colony-forming assays. D, The cell growth curve of IDH3b-overexpressed cell and control group. E, Xenograft tumor formation from stable IDH3b-overexpressed cells and control cells. Representative images (left) and quantitative analysis (right) of xenograft tumor volumes. Error bars indicate SEM of three independent experiments. , P < 0.05; , P < 0.01; , P < 0.001. Scale bar, 5 mm.

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Figure 4. IDH3b-dependent a-KG generation accounts for improving cell proliferation. A, The proliferation curve of cells that were supplied with a-KG. B, KYSE410 cells were supplied with a-KG for 4 days and the cell-cycle distribution was analyzed by flow cytometry. C, KYSE410 cells were supplied with a-KG for 4 days and then incubated with Edu (50 mmol/L) for 1.5 hours. At least 300 cells were examined per slide. D, The colony-forming ability of KYSE410 cells supplied with a-KG was detected. E, The proliferation curve of IDH3b knockdown cells and control cells with or without a-KG in KYSE410. F, The cell-cycle profile of IDH3b knockdown cells and control cells with or without a-KG in KYSE410. G, The Edu incorporation of IDH3b knockdown cells and control cells with or without a-KG in KYSE410. Error bars indicate SEM of three independent experiments. , P < 0.05; , P < 0.01; , P < 0.001. Scale bar, 5 mm. ns, nonsignificant.

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Figure 5. IDH3b enhances glucose uptake and PFKFB3 level. A and B, Equal numbers of KYSE30 cells and KYSE410 cells were collected after transfection for glucose consumption determination. C, PFKFB3 protein level in IDH3b-overexpressed and IDH3b knockdown cells was determined by immunoblotting. D, Nuclear and cytoplasmic PFKFB3 level in IDH3b-overexpressed KYSE30 cells was determined by immunoblotting. E, The subcellular distribution of PFKFB3 in IDH3b- overexpressed KYSE30 cells is shown by immunoﬂuorescence, and the relative ﬂuorescence intensity in 50 cells was calculated by ImageJ. Error bars indicate SEM of three independent experiments. , P < 0.001.

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led to an induction of both nuclear and cytoplasmic PFKFB3 significantly correlated with poor survival among patients with (Fig. 5D and E; Supplementary Fig. S8). As previous studies ESCC, indicating that IDH3b-dependent metabolic alteration reported that IDH3a might exert its roles not only in TCA cycle contributes to ESCC progression and IDH3b can serve as a but also glycolysis (21, 22), the close relationship between IDH3b promising prognostic molecular marker. and PFKFB3 shown here further supports a critical role of IDH3b Metabolic network is integrated to cell-cycle progression. Cell- in glycolysis. cycle regulators such as cyclins, CDKs, or E2F factors had pro- found impact on metabolism in numerous human cancers (38). IDH3b overexpression is significantly correlated with poor Among those well-studied regulators, APC/C is quite fascinating survival among patients with ESCC for its special role in affecting glycolysis and glutaminolysis (39). Copy number alterations often lead to metabolism reprogram- With great interests, we wondered whether APC/C would take part ming during tumor progression (36). IDH3b was amplified in in TCA cycle pathways. On the basis of the central importance of ESCC and we found the DNA copy number of IDH3b was TCA cycle, IDH, succinate dehydrogenase, and fumarate hydratase positively correlated with its mRNA expression in TCGA cohort in TCA cycle have been shown to be directly responsible for the (Fig. 6A, P < 0.001). Next, we performed IHC assay to evaluate the cancer initiation and progression (40). From the APC/C substrate expression level of IDH3b in 340 paired samples of ESCC tissues, candidates screened under rigorous criteria, we conducted further matched adjacent normal tissues, and lymph node metastasis research of IDH3b in ESCC. We validated that IDH3b was APC/C tissues (Supplementary Table S3). Consistently, IDH3b was substrate as expected. Then, we observed that IDH3b accumulated expressed at higher levels in ESCC tumor tissues than in adjacent in late G1 phase to early S-phase but remained a low level during normal tissue, and at higher levels in metastatic lymph nodes mitosis phase. However, we only confirmed CDH1, not CDC20, (Fig. 6B and C, P < 0.001). The 5-year survival rate of IDH3b- that degraded IDH3b in early G1 phase. Besides, the S-phase positive expression group was substantially lower compared with kinase-associated protein 1 (SKP1)–cullin 1–F-box protein (SCF), the IDH3b-negative expression group (40.7% vs. 57.6%). We SKP2, and b-transducin repeat-containing protein (b-TRCP) com- further explored the association between IDH3b expression level plexes are widely studied E3 ubiquitin ligases in cell cycle as and clinicopathologic features of patients with ESCC. Statistical well (41). Thus, there might be other regulations that exist to affect analyses revealed that IDH3b expression level was significantly IDH3b protein level in mitosis phase. positive correlated with family history of cancer (P ¼ Biochemical evidence indicated that IDH1 and IDH2 operated 0.003, Table 1). Importantly, IDH3b overexpression was strongly in the reverse direction in TCA cycle and synthesized D-threo- associated with poor overall survival (OS; P ¼ 0.011, Kaplan– isocitrate, while IDH3 operated in the forward direction and Meier survival analysis and log-rank test, Fig. 6D) and disease-free synthesized a-KG (42). Accordingly, we examined the intracel- survival (DFS) of ESCC patients (P ¼ 0.038, Kaplan–Meier lular a-KG level and found it was positive correlated with IDH3b survival analysis and log-rank test, Fig. 6E). The median OS time level in ESCC cells. Furthermore, we demonstrated that a-KG and DFS time in the IDH3b-positive expression group were 38 played a new role in promoting G1–S transition and cell growth. and 28 months, respectively, while 106 and 76 months, respec- However, previous study reported that IDH3a induced HIF-1– tively, in the IDH3b-negative expression group. Multivariate Cox mediated metabolic reprogramming by decreasing a-KG levels in regression survival analysis adjusting for age, T stage, LNM, HeLa cells (21). Because both IDH3a and IDH3b are the subunits differentiation, gender, tumor embolus, family history, hematog- of IDH3, the different a-KG level under similar treatment need enous metastasis, and IDH3b level consistently reported strong further discussion. In that work, the IDH3a-induced a-KG reduc- correlation between IDH3b-positive expression and shorter OS (P tion was identified as glycolysis independent, but we observed ¼ 0.029, HR ¼ 1.484, 95% CI, 1.041–2.142, Fig. 6F). However, it higher glucose uptake and lifted PFKFB3 level after upregulating did not come to a significant correlation with DFS (P > 0.05, HR ¼ IDH3b. As views renewed that glucose and lactate can be a 1.232; 95% CI 0.877–1.730, Fig. 6G), showing that IDH3b replenishment source for TCA cycle (43, 44), the increased a-KG expression was an independent prognostic factor for outcome in level may be due to the enhanced glycolytic flux. In addition, we ESCC. Taken together, these findings strongly suggest that IDH3b noted that IDH3b triggered accelerated S-phase entry, in which the is dysregulated in ESCC and holds promise as a prognostic GLS1 was accumulated (45). To meet heightened demands for marker. intermediates followed by increasing proliferation, the accumulated GLS1 may indirectly account for raising a-KG to feed TCA cycle. Discussion Although the nuclear and cytoplasmic PFKFB3 were increased Our findings confirmed that IDH3b was a new APC/C-CDH1 after IDH3b overexpression, it is not clear whether such induction substrate and expressed in a cell-cycle–dependent manner, which is directly regulated by IDH3b, or it is a secondary effect of demonstrated a new cross-talk regulation between cell cycle and triggering G1–S transition. Therefore, the molecular mechanism TCA cycle. Given that the relationship between cellular metabo- of IDH3b and PFKFB3 needs to be elucidated in the further study. lism and cell-cycle machinery was bidirectional (37), here, we not However, increased PFKFB3 and lifted glucose uptake would only displayed the regulation of APC/C on IDH3b, but also contribute to glycolysis and produce more ATP, while overexpres- revealed the ability of IDH3b to promote G1–S transition, result- sion of IDH3b in TCA may benefit a lot in biosynthesis. The ing in increased cell proliferation in vitro and in vivo. These results correlation provided clues that different metabolic pathways may presented a new two-way regulation between cell cycle and TCA cooperate on tumorigenesis and progression. cycle. In addition, IDH3b overexpression resulted in elevation of Metabolic reprogramming is one of the critical characteristics of PFKFB3 protein level. These observations raised a novel concept cancer and metabolic alterations that has been substantiated in that there existed regulation between key enzymes in TCA cycle cancer cells for decades (46), so the abnormal metabolic activities and glycolysis. Importantly, high expression level of IDH3b was in ESCC may lead to tumorigenesis. Integrative metabolic

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Figure 6. IDH3b overexpression is associated with poor survival in ESCC. A, The DNA copy number of IDH3b positively correlated with its mRNA expression. B, Micrographs of IHC against IDH3b in ESCC tissues (T), matched adjacent normal tissues (N), and metastatic lymph nodes (LNM). C, The frequency of IDH3b expression levels in ESCC tissues, matched adjacent normal tissues, and metastatic lymph nodes. D and E, Kaplan–Meier survival analysis of all patients with ESCC stratiﬁed by IDH3b expression level shows poor OS (D) and poor disease-free survival (E). F and G, Multivariate Cox regression survival analysis of associations between survival and various clinicopathologic factors. HRs and 95% CIs are plotted for each variable. Left, OS (F); right, disease-free survival (G). Scale bar, 200 mm. , P < 0.05; , P < 0.001.

pathway analysis of genes and metabolites was performed in detailed investigations also put forward enzymes that were abnor- ESCC to locate 39 signiﬁcant subpathway regions, including mally expressed in ESCC, such as choline kinase (48), glucose amino acid metabolism, glucose metabolism, glutathione metab- transporter isoform 1 (GLUT1; ref. 49), IDH2 (50), were closely olism, lipid metabolism, TCA cycle, and so on (47). Several related with cancer progression.

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Table 1. Association of IDH3b expression with clinicopathologic features of 340 overexpression may act as a significant biomarker for ESCC ESCC patients prognosis. IDH3b Expression Clinicopathologic Total cases Positive Negative fl features (%) (%) (%) P Disclosure of Potential Con icts of Interest fl Gender No potential con icts of interest were disclosed. Male 278 (81.8) 207 (74.5) 71 (25.5) 0.112 Female 62 (18.2) 40 (64.5) 22 (35.5) Authors' Contributions Age Conception and design: Q. Wu, W. Zhang, Y. Song, Qi-Min Zhan 60 190 (55.9) 132 (69.5) 58 (30.5) 0.14 Development of methodology: Q. Wu, W. Zhang, Qi-Min Zhan >60 150 (44.1) 115 (76.7) 35 (23.3) Acquisition of data (provided animals, acquired and managed patients, Lymph node metastasis provided facilities, etc.): Q. Wu, W. Zhang, L. Xue, Y. Wang Yes 171 (50.3) 126 (73.7) 45 (26.3) 0.666 Analysis and interpretation of data (e.g., statistical analysis, biostatistics, No 169 (49.7) 121 (71.6) 48 (28.4) computational analysis): Q. Wu, W. Zhang, Y. Wang Family history Writing, review, and/or revision of the manuscript: Q. Wu, W. Zhang, Qi-Min Yes 60 (17.8) 53 (88.3) 7 (11.7) 0.003 Zhan No 278 (82.2) 193 (69.4) 85 (30.6) Administrative, technical, or material support (i.e., reporting or organizing Differentiation data, constructing databases): Q. Wu, W. Zhang, M. Fu, L. Ma High/median 247 (75.3) 178 (72.1) 69 (27.9) 0.569 Study supervision: Y. Song, Qi-Min Zhan Low 81 (24.7) 61 (75.3) 20 (24.7) T_Stage T2 88 (25.9) 67 (76.1) 21 (23.9) 0.394 Acknowledgments T3 252 (74.1) 180 (71.4) 72 (28.6) We are grateful to Mr. Jiedan Chen and Mr. Lin Li for bioinformatic support. Hematogenous metastasis This work was supported by the National Natural Science Foundation of China Yes 60 (17.6) 39 (65.0) 21 (35.0) 0.143 (81490753 to Q. Zhan), the National Basic Research Program of China No 280 (82.4) 208 (74.3) 72 (25.7) (2015CB553904 to Q. Zhan), China Postdoctoral Science Foundation (2017M620010 to W. Zhang), National Postdoctoral Program for Innovative Talent (to W. Zhang), the National Key Research and Development Program of In this study, we demonstrated that IDH3b contributed to China (no. 2016YFA0500303 to Y. Song), and CAMS Innovation Fund for malignant phenotypes of ESCC cells, including facilitating cell Medical Sciences (CIFMS; grant no. 2016-I2M-1-001 to Y. Song). proliferation, colony formation, cell invasiveness, and mobility The costs of publication of this article were defrayed in part by the payment of and xenograft tumor formation. We also found that IDH3b was advertisement fi fi page charges. This article must therefore be hereby marked in frequently ampli ed in ESCC and the protein level was signi - accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cantly higher in ESCC tissues than in adjacent normal tissue. Statistical analysis indicated that high IDH3b expression was Received August 1, 2018; revised December 20, 2018; accepted April 29, 2019; tightly associated with poor OS and DFS. In conclusion, IDH3b published first May 3, 2019.

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APC/C-CDH1−Regulated IDH3β Coordinates with the Cell Cycle to Promote Cell Proliferation

Qingnan Wu, Weimin Zhang, Liyan Xue, et al.

Cancer Res 2019;79:3281-3293. Published OnlineFirst May 3, 2019.

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