ldha in neuroblastoma is associated with poor outcome and its … · clin cancer res; 24(22);...

Translational Cancer Mechanisms and Therapy

LDHA in Neuroblastoma Is Associated withPoor Outcome and Its Depletion DecreasesNeuroblastoma Growth Independent ofAerobic GlycolysisCarmen Dorneburg1, Matthias Fischer2, Thomas F.E. Barth3,Wolfgang Mueller-Klieser4,Barbara Hero2, Judith Gecht2, Daniel R. Carter5, Katleen de Preter6, Benjamin Mayer7,Lisa Christner1, Frank Speleman6, Glenn M. Marshall5,8, Klaus-Michael Debatin1, andChristian Beltinger1

Abstract

Purpose: To investigate whether lactate dehydrogenase A(LDHA), an important component of the LDH tetramer crucialfor aerobic glycolysis, is associated with patient outcome andconstitutes a therapeutic target in neuroblastoma (NB).

Experimental Design: Expression of LDHA mRNA andprotein was determined in 709 and 110 NB patient samples,respectively, and correlated with survival and risk factors.LDHA and LDHB were depleted in human NB cell lines byCRISPR/Cas9 and shRNA, respectively, and aerobic glycolysis,clonogenicity, and tumorigenicity were determined. Expres-sion of LDHA in relation to MYCN was measured in NB celllines and in the TH-MYCN NB mouse model.

Results: Expression of LDHA, both on the mRNA and theprotein level, was significantly and independently associat-ed with decreased patient survival. Predominant cyto-plasmic localization of LDHA protein was associated with

poor outcome. Amplification and expression of MYCN didnot correlate with expression of LDHA in NB cell lines orTH-MYCN mice, respectively. Knockout of LDHA inhibitedclonogenicity, tumorigenicity, and tumor growth withoutabolishing LDH activity or significantly decreasing aerobicglycolysis. Concomitant depletion of LDHA and the isoformLDHB ablated clonogenicity while not abrogating LDHactivity or decreasing aerobic glycolysis. The isoform LDHCwas not expressed.

Conclusions: High expression of LDHA is independentlyassociated with outcome of NB, and NB cells can be inhibitedby depletion of LDHA or LDHB. This inhibition appears tobe unrelated to LDH activity and aerobic glycolysis. Thus,investigations of inhibitory mechanisms beyond attenuationof aerobic glycolysis are warranted, both in NB and normalcells. Clin Cancer Res; 24(22); 5772–83. �2018 AACR.

IntroductionNeuroblastoma (NB) is the most common extracranial solid

tumor of childhood. The transcription factor MYCN, whose geneis often amplified in poor prognosis NB (1, 2), has long beenknown to convey a poor prognosis by inducing diverse target

genes (3). Although the poor prognosis of high-risk patients hasimproved in the last decades, many patients still die from theirdisease (4, 5). Therefore, novel prognostic markers and therapeu-tic targets are needed.

Aerobic glycolysis, also known as the Warburg effect, is ahallmark of cancers, including NB (6–8). Aerobic glycolysisincreases the provision of metabolic building blocks and rendersthe tumor microenvironment permissive for tumor growth, bothof which endows cancer cells with a growth advantage despite theenergetic inefficiency of aerobic compared with anaerobic glycol-ysis (9). As aerobic glycolysis occurs in cancer but not in normalcells, it constitutes a promising therapeutic target. Along this line,inhibition of aerobic glycolysis has been shown to decreasegrowth of cancer cells, including NB (10–12).

Lactate dehydrogenase (LDH) is a tetrameric enzyme com-posed of either lactate dehydrogenase A (LDHA) or B (LDHB)subunits, or combinations thereof, or LDHC (13). LDHA isutilized by cancer cells to bypass oxidative phosphorylationby reducing pyruvate to lactate (9). This diverts metabolicprecursors of pyruvate into the pentose phosphate pathway,which supplies metabolic building blocks for cancer cellgrowth (9). Elevated extracellular lactate levels enhance tumorangiogenesis, immune escape, and additional parameters ofthe tumor microenvironment conducive for tumor growth

1Department of Pediatrics and Adolescent Medicine, University Medical CenterUlm, Ulm, Germany. 2Department of Pediatric Oncology and Hematology,Children's Hospital, University of Cologne, Albertus-Magnus-Platz, K€oln,Germany. 3Institute of Pathology, University Medical Center Ulm, Ulm, Germany.4Institute of Pathophysiology, University Mainz, Germany. 5Children's CancerInstitute Australia, Lowy Cancer Centre, University of New South Wales, NSW,Sydney, Australia. 6Center for Medical Genetics (CMGG), Ghent University,Ghent, Belgium. 7Institute of Epidemiology and Medical Biometry, Ulm Univer-sity, Ulm, Germany. 8Kids Cancer Centre, Sydney Children's Hospital, Sydney,Australia.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Christian Beltinger, University Medical Center Ulm,Eythstr. 24, Ulm 89075, Germany. Phone: 731-500-57032; Fax: 731-500-57042; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-2578

�2018 American Association for Cancer Research.

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(14–17). LDHA is known to be a target gene of c-MYC (18) andof hypoxia-inducible factor-1a (HIF-1a; ref. 19), and may be atarget of MYCN (20), consistent with a role of LDHA in tumormaintenance. Along this line, overexpression of LDHA is asso-ciated with unfavorable prognosis of several cancers (21, 22)and with resistance to radiotherapy (23). Conversely, inhibi-tion of LDHA can reduce cancer progression (24–29). Similarto LDHA, LDHB is also associated with aggressive cancerphenotypes (30, 31). Targeting LDHB decreases cancer cellproliferation (30, 31) and autophagy (32).

As little is known about aerobic glycolysis, LDHA, and LDHB inNB, we investigated these aspects in detail. In a large patientcohort, we show that LDHA mRNA and protein expressionand predominant cytoplasmic localization of LDHA protein areassociated with increased tumor aggressiveness and decreasedpatient survival. Knocking out LDHA and knocking down LDHBdecreased malignant characteristics of NB cells. Surprisingly,aerobic glycolysis remained unaffected.

Materials and MethodsAnalysis of LDHA mRNA expression, patient outcome and riskfactors

Clinically annotated gene expression profiles previously gen-erated from NB patients using a 44k oligonucleotide array (33)were analyzed for LDHA mRNA expression, as described inSupplementary Information.

Analysis of LDHA protein expression, patient outcome and riskfactors

The Neuroblastoma Tumor Bank in Cologne, Germany, andthePediatric TumorRegistry inKiel, Germany, provided apanel oftumors from initial diagnosis that are described in SupplementaryInformation.

The formalin-fixed and paraffin-embeddedNB specimens weresubjected to H&E and immunohistochemical staining for LDHAaccording to standardmethods. To quantify LDHA protein, slideswere analyzed using a Keyence microscope BZ-9000 and Keyenceimage analysis software. The LDHA-positive tumor area wasdetermined and expressed as the percentage of total tumor areaon the slide. For subcellular investigation, the percentages of cellswith LDHA-positive cytoplasm and LDHA-positive nuclei in eachsample were determined by a pathologist blinded to patient data.Tumors with �5% or <5% positive cells in the respective cellularcompartment were categorized as positive or negative, respective-ly. Statistical analysiswas performed as detailed in SupplementaryInformation.

LDHA in the TH-MYCN NB progression modelThe TH-MYCN NB progression model has been described

elsewhere (34). Briefly,TH-MYCNþ/þmicewere sacrificedatweeks1 and 2 postnatally to isolate sympathetic ganglia containing fociof neuroblast hyperplasia, and at week 6 to harvest advanced NBtumors (35). Sympathetic ganglia from TH-MYCN�/� (wild-type)mice atweek1, 2, and6wereusedas control for expression changesduring normal development.Murine total RNAwas isolated usingthe RNeasy Mini Kit (Qiagen). The samples were profiled onAgilent SurePrint G3 Gene Expression Microarrays accordingto the manufacturer's protocol. Data were summarized and nor-malizedwith the vsnmethod (36) in theR statistical programminglanguage using the limma package. Linear regression analysis wasperformed to evaluate the differential temporal expression patternin ganglia from wild-type mice and ganglia and tumors fromtransgenic mice.

Generation of LDHA knockout NB cellsThree different sgRNAs (Supplementary Information) were

cloned intoGeneArt CRISPRNuclease Vector (Life Technologies).NB cells were transiently transfected with sgRNA plasmids, sortedand seeded as single cells. Single cell clones were expanded andDNA was isolated using DirectPCR Lysis Reagent Cell (Peqlab).Exon 2 of LDHA was PCR-amplified (Supplementary Table S1)and Sanger-sequenced. Sequences were analyzed for indels in thetarget region and for absence of off-target mutations in the fivegenes most likely to harbor such mutations. The clones wereclassified as having LDHA wild-type sequence or homozygousknockout sequences, that is indels leading to premature stopcodons. Knockout of LDHAwas verified byWestern blot analysis.

Knockdown of LDHB by shRNANB cells were stable transduced with three LDHB shRNA

lentiviruses and one nonsilencing control virus at a MOI of20 (TRIPZ, Dharmacon; #RHS4740-EG3945 and #RHS4743)according to the manufacturer's protocol. After stable selectionwith puromycin, cells were treated with 1 mg/mL doxycycline(Sigma) to induce shRNA expression. To determine knockdownefficiency Western blots for LDHB protein were performed.

Glucose and lactate determination in vitroGlucose and lactate levels in cell culture medium were deter-

mined using colorimetric glucose and lactate assays (BioVisionkits K606-100 and K627-100) according to the manufacturer'sinstructions. Briefly, NB cells were seeded in six-well plates andafter indicated time points medium was collected and deprotei-nized. Subsequently, 50 mL of diluted samples were used tomeasure lactate and glucose concentrations using a BioTek ELISAreader.

LDH activity assayThe LDH activity of NB cells was determined according to the

manufacturer's protocol (Sigma;MAK066). A total of 1 to 2� 106

cells were suspended with 500 mL LDH assay buffer and centri-fuged with 10,000 x g at 4�C for 15 minutes. LDH activity wasmeasured in supernatants and cell lysates at 450 nm using aBioTek ELISA reader.

Animal experimentsSix to eight weeks old male and female immunodeficient

RAG2�/� common gamma chain�/� (RAG2�/�/cgc�/�) mice

Translational Relevance

Being independently associated with poor outcome, LDHAexpression could be incorporated into signatures to assess ifLDHA expression improves risk prediction in NB. LDHA mayalso be considered as a therapeutic target in NB if the growth-controlling effects of LDHA inhibition can be improved bycombining itwith additional therapeutic approaches. The datasuggest that for this purpose, targetingmechanisms other thanaerobic glycolysis should be considered.

LDHA and Aerobic Glycolysis in Neuroblastoma

www.aacrjournals.org Clin Cancer Res; 24(22) November 15, 2018 5773




bred in the Animal ResearchCenter ofUlmUniversity andhousedin sterile isolators were used. NB cell line clones KELLY, LAN-5,SK-N-AS, and SK-N-BE(2)C were chosen for injection. A total of5 � 105 viable cells (1 � 106 of SK-N-AS cells) in 25% highconcentration matrigelTM (BD Biosciences) in DMEM/F12(Gibco) were subcutaneously injected. Mice were monitoredregularly and tumor size was measured using a caliper [v ¼1/2(W � W � H)]. Doubling time was calculated from tumorvolume over time. Mice were sacrificed when tumors reached 1.5cm in diameter or when tumors penetrated the skin. All experi-ments were done according to state and institutional guidelinesfor the care and protection of research animals.

Glucose, lactate, and ATP determinations in vivoGlucose, lactate, and ATP content in tumor sections were

measured by induced metabolic bioluminescence imaging, asdescribed previously (37). In brief, cryostat sections of shock-frozen tissue specimens were immersed into an enzyme solution.Defined increase of temperaturemade the tissue sectionsmelt andallowed for enzymatic reactions to take place, eventually leadingto emission of light. The light was registered with a precisionmicroscope (Axiophot) and an ultrasensitive video system(Ancor) to calculate local metabolite content in micromoles pergram (mmol/g) of tissue. Additional information is provided inSupplementary Material and Methods.

ResultsHigh expression of LDHA mRNA is independently associatedwith poor outcome

We assessed association of LDHA transcripts with survival andwith established risk factors of NB. To this end, we performed insilico analysis in a large number of clinically annotated NB. HighLDHA expression was significantly associated with markedlylower overall and event-free survival (Fig. 1A; SupplementaryFig. S1), independent of MYCN, age, and stage (Fig. 1B; Supple-mentary Table S2). Increased levels of LDHA mRNA were signif-icantly associated with MYCN amplification, increased age,advanced stage, and undifferentiated histology (Fig. 1C). Takentogether, in this study, increased expressionof LDHAmRNA inNBwas independently associated with poor outcome and correlatedwith established risk factors.

High LDHA protein levels and increased cytoplasmic anddecreased nuclear LDHA protein are significantly associatedwith poor outcome

To investigate whether protein expression of LDHA in NB isassociated with outcome and established risk factors of NB, weperformed immunohistochemistry (IHC) of LDHA in 110 clin-ically annotated patient samples. For LDHA protein quantifica-tion, the fraction of LDHA-positive tumor area of total tumor areawas determined on the slides (Fig. 2A). Kaplan–Meier analysisshowed a marked association of LDHA protein expression withdecreased overall and event-free patient survival (Fig. 2A), whichwas independent ofMYCN and age for overall but not for event-free survival (Fig. 2B; Supplementary Table S3). Validation of theresults in additional cohorts is required. Increased expression ofLDHA protein was significantly associated with amplification ofMYCN, increased age, and advanced stage (Fig. 2C).

Given its known function, we hypothesized that LDH com-partmentalized into the cytoplasm but not the nucleus would be

negatively associated with survival. Indeed, this compartmental-ization (Fig. 2D) appeared to be associated with decreased overallsurvival (Fig. 2E).

More MYCN-amplified, advanced stage, and undifferentiatedNB contained cells with cytoplasmic LDHA (Fig. 2F). The increaseof cytoplasmic LDHA in NB of older patients was small.

Thus, in this study, high LDHA protein levels were associatedwith poor outcome of NB and correlated with risk factors of NB.

Expression of LDHA in NB of TH-MYCNmice is not induced byMYCN

Although LDHA is a known target gene of c-MYC (18), it isunknown whether it is also a target gene of MYCN. To start toaddress this question, we first investigated LDHA mRNA expres-sion during the development of NB from tumor-prone gangliaat 2 weeks of age to tumors at 6 weeks of age in TH-MYCN trans-genic mice by in silico analysis (35). Although expression ofODC1, a bona fide target gene of MYCN, markedly increasedduring neuroblastomagenesis compared with developing gangliaof wild-type mice, expression of LDHA did not (Fig. 3A). Thisshows that LDHA in NB of TH-MYCN mice is not induced byexpression of MYCN.

Expression of LDHA and LDHB in human NB cell lines is notincreased when MYCN is amplified

To address the question whether MYCN amplification is asso-ciated with enhanced LDHA expression, we determined LDHAtranscript and protein levels in 10 NB cell lines and 2 derivativeswith single and increased copy number of MYCN (Fig. 3B). Nodifference was detected. Interestingly, SK-N-BE(2)C cells werecompletely devoid of LDHA. As LDHA, LDHB was not differen-tially expressed in the amplified and nonamplified NB cell lines(Fig. 3B).

ForcedoverexpressionofMYCNdoesnot increase expressionofLDHA and the regulator HIF-1a in human SH-EP NB cells

Next, we investigated whether acute exposure of NB cells toMYCN would induce LDHA. To this end, we triggered nucleartranslocation of MYCN-ER in the SH-EP MYCN-ER cell line byadding tamoxifen (38). Although the MYCN target gene ODC1was robustly induced LDHA expression was not (Fig. 3C). MYCNis known to induce key Warburg effect enzymes, either directly orvia HIF-1a. We therefore analyzed whether MYCN and HIF-1acooperate in inducing key Warburg effect enzymes. As HIF-1aprotein is only present when stabilized by hypoxic conditions, wesimulated the effect of hypoxia on HIF-1a by stabilizing HIF-1awith deferoxamine. MYCN did not significantly increase HIF-1aprotein (Supplementary Fig. S2A) normRNA levels of LDHAor ofother Warburg enzymes examined in SH-EP MYCN-ER cells(Supplementary Fig. S2B). Deferoxamine significantly increasedmRNA of Glut1, PDK1, and LDHA (Supplementary Fig. S2B).Of note,MYCNandHIF-1a did not cooperate in the transcriptionof the Warburg enzymes investigated.

Taken together, the data do not support the notion thatincreasedMYCN, per se or together with HIF-1a, enhances expres-sion of LDHA and other key enzymes of aerobic glycolysis in theNB cell lines investigated.

LDHA depletion decreases aggressiveness of NB cellsTo investigate the relevance of LDHA for aggressiveness of NB

cells, LDHAwas knocked out in SK-N-AS and KELLY NB cell lines

Dorneburg et al.

Clin Cancer Res; 24(22) November 15, 2018 Clinical Cancer Research5774




using CRISPR/Cas9. SK-N-AS and KELLY cell lines were chosenbecause they are representative of MYCN nonamplified andMYCN-amplified NB cell lines, respectively. Clones expandedfrom single cells after transfection of CRISPR/Cas9 and LDHAsgRNAs were Sanger-sequenced and probed for LDHA protein byWestern blotting. In all clones without LDHA protein (Fig. 4A)and with unambiguous sequence analysis LDHA was homozy-

gously knocked out (data not shown), whereas all clones expres-sing LDHA were wild type. Homozygous knockout clones andwild-type clones were randomly chosen for further experiments.

Knockout of LDHA significantly decreased clonogenicity ofboth SK-N-AS and KELLY NB cells (Fig. 4B).

To determine the influence of LDHA on tumorigenicity andin vivo growth of NB cells, LDHA-knockout and wild-type clones

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Figure 1.

Increased LDHA mRNA expression in patient NB is independently associated with poor outcome. A, High transcript levels of LDHA in NB are significantly associatedwith decreased survival of patients. Overall and event-free survival of 481 patients with NB according to LDHA transcript levels. The prognostic LDHA cutoffhad been determined in a training set bymaximally selected log-rank statistic. B, The association of LDHAmRNAwith survival is independent of known risk factors ofNB. Cox proportional hazard regression (HR) analyses using the optimal prognostic cutoff expression for LDHA were performed. LDHA expression and the riskfactors MYCN status (na, nonamplified; a, amplified), age, and tumor stage were analyzed. CI, confidence interval. C, Increased LDHA transcript levels are associatedwith risk factors of NB. LDHA mRNA levels of the 709 patients were analyzed depending on risk factors. Data are presented as box plots; na, nonamplified MYCN;a, MYCN-amplified; n, number of samples. P values were calculated using the Mann–Whitney test, except for the INPC, where the Kruskal–Wallis test was used;�� , P < 0.001; �� , P < 0.01.






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Figure 2.

Increased expression of LDHA protein in patient NB is associated with poor outcome. A, High LDHA protein expression in NB is significantly associated withdecreased survival of patients. Overall and event-free survival of 110 patients with NB depending on LDHA protein expression, as determined by IHC (left). Statisticalanalysis by log-rank test. The panels on the right show a representative LDHA-stained tumor slide and its image analysis. (Continued on the following page.)

Dorneburg et al.





of SK-N-AS and KELLY cells were transplanted subcutaneouslyinto Rag2�/� cgc�/� mice. LDHA depletion decreased tumorincidence in SK-N-AS cells and prolonged tumor latency (Fig.4C) and tumor-doubling time (Fig. 4D) of both SK-N-AS andKELLY NB cells. Together, these results show that knockout ofLDHA decreases aggressiveness of NB cells.

LDHA is dispensable for the Warburg effect in NB cellsTo assess the metabolic effects of LDHA depletion in NB cell

lines, we determined LDH activity of LDHA-depleted and -repleteNB cells. Although LDH activity was reduced in cells with knock-out of LDHA residual LDH activity was maintained (Fig. 5A). Ofnote, glucose consumption and lactate production were notsignificantly altered in LDHA-depleted cells under aerobic cultureconditions (Fig. 5A).

To determine the situation in vivo, SK-N-AS, LAN-5, and SK-N-BE(2)C cells were subcutaneously transplanted into immunode-ficient mice and tumor tissues were stained for LDHA. WhileLDHAwas highly expressed in SK-N-AS and LAN-5 tumors, SK-N-BE(2)C cell tumors were completely devoid of LDHA (Fig. 5B).This was not due to mutations in the coding sequence of LDHA(data not shown). Tumors derived from SK-N-AS, LAN-5, and SK-N-BE(2)C cells were analyzed for themetabolites glucose, lactate,and ATP (Fig. 5C). Despite the complete absence of LDHA, SK-N-BE(2)C tumors consumed glucose and generated lactate similar tothe LDHA-replete NB cell lines SK-N-AS and LAN-5. SK-N-BE(2)Ctumors also generated similar amounts of ATP as the LDHA-replete LAN-5 tumors. These data show that NB cells completelylacking LDHA can still maintain aerobic glycolysis.

Concomitant depletionof LDHAandLDHB inNBcells doesnotabrogate LDH activity and the Warburg effect while abolishingclonogenic growth

LDHactivity is supposed to be essential for aerobic glycolysis intumor cells. We reasoned that LDHB could rescue LDH activitywhen LDHA is depleted. Seemingly supporting this notion, wefound that LDHB was clearly and invariably expressed in NB cellclones both replete with and depleted of LDHA (Fig. 6A). Todirectly assess the relevance of LDHB in NB cells, doxycycline-dependent inducible short-hairpin RNAs (shRNA) were used toknockdown LDHB. LDHBwas strongly reduced in both wild-typeand LDHA-depleted clones whereas LDHC was not expressed(Fig. 6B). Expression of LDHCmRNAwas very low inNB cell linesand patient NB (Supplementary Fig. S3). Knockdown of LDHBdid not decrease LDH activity in SK-N-AS cells and only margin-ally in KELLY cells (Fig. 6C). Of note, combined depletion ofLDHA and LDHB did not abrogate LDH activity (Fig. 6C).

Neither depletion of LDHB alone nor in combination withLDHA depletion affected glucose consumption and lactateproduction (Fig. 6C).

To assess the effect of combined depletion of LDHB and LDHAon malignant behavior in vitro, we investigated clonogenicgrowth. Depletion of LDHB alone decreased clonogenic growthof SK-N-AS cells significantly and of KELLY cells profoundly (Fig.6D). Combined depletion of LDHA and LDHB abrogated clono-genic growth in both cell lines (Fig. 6D).

Taken together, simultaneous depletion of LDHB and LDHA inSK-N-AS and KELLY cells does not decrease aerobic glycolysiswhile abolishing clonogenicity.

DiscussionThis paper shows that increased expression of LDHA is asso-

ciated with decreased survival in NB. The data support the notionthat LDHA and its isoform LDHB contribute to aggressiveness ofNB cells while being dispensable for aerobic glycolysis. This iscompatible with nonmetabolic protumor functions of LDHA andLDHB in NB. Additional studies are necessary to validate thesenovel results.

We have shown in this large cohort of NB patients thatincreased LDHA mRNA was associated with markedly decreasedoverall survival while correlating with parameters of aggressivedisease, that is amplification of MYCN, older age, stage 4, andundifferentiated histology. Similarly, an increased fraction oftumor cells expressing LDHA protein was associated with pooroutcome in NB. The latter supports the notion that enhancedexpression of LDHA is not an epiphenomenon andmay facilitateclinical application, as immunohistochemical analysis is a stan-dardmethod in the diagnostic work-up of NB. Along this line, wefound that subcellular detection of LDHA protein might provideadditional prognostic information, as cytoplasmic but not nucle-ar expression of LDHA appeared to be associated with poorersurvival. Future prospective validation of this conclusion iswarranted.

At first glance, the association of increased overall and cyto-plasmic expression of LDHAwith prognosis of NBmay be readilyexplained by the role of LDHA in the Warburg effect. Along thisline of argument, nuclear LDHAwould represent enzyme seques-tered from its cytoplasmic compartment, where aerobic glycolysisfunctions of LDHA are located. However, we provide evidencethat LDHA is not necessary for aerobic glycolysis in the NB celllines analyzed. This raises the question whether LDHA has func-tions unrelated to aerobic glycolysis. Indeed, LDHA has beenreported to regulate mRNA stability of nonglycolytic genes (39)

(Continued.) Bars equal 50 mm. The LDHA-positive tumor area was expressed as the percentage of total tumor area and used as a surrogate for LDHAprotein expression. B, The association of LDHA protein expressionwith survival is independent of known risk factors of NB. Cox proportional hazard regression (HR)analyses for LDHA protein expression, MYCN status, and age are shown. C, Increased LDHA protein levels are associated with risk factors of NB. TheNBpatient samples stained for LDHAwere analyzed in relation to risk factors. Data are presented as box plots. na, nonamplifiedMYCN; a,MYCN-amplified; n, numberof samples. P values were calculated using the Mann–Whitney test, except for INPC, where the Kruskal–Wallis test was used. �� , P < 0.01; �, P < 0.05; n.s., notsignificant. D, LDHA in human NB is located in cytoplasm and nuclei. The NB stained for LDHA was analyzed using higher magnification. Representative images oftumor sections with LDHA-positive cytoplasm (top) and LDHA-positive nuclei (bottom) are shown. Bars equal 50 mm. E, Increased and decreased numbersof cells with cytoplasmic and nuclear LDHA, respectively, are associated with decreased overall survival of patients with NB. In the NB stained for LDHA, thepercentages of cytoplasmic-positive and nuclear-positive cells within each tumor were determined. Tumors with �5% or <5% positive cells in the respectivecellular compartment were categorized as positive or negative, respectively. Kaplan–Meier survival analysis of the NB patients according to the subcellulardistribution of LDHA is shown. Statistical analysis by log-rank test. F, Increased number of cells with cytoplasmic LDHA in NB is associated with risk factors.The fraction of tumors with LDHA in the cytoplasm was analyzed for association with MYCN status (na, nonamplified; a, amplified), age, stage, and differentiation(diff., differentiated; p., poorly differentiated; undiff., undifferentiated); CI, 95% confidence interval.






and may impact on replication independent of aerobic glycolysis(40). It is tempting to speculate that nonglycolytic targets ofLDHA, both in the cytoplasm and the nucleus, include some thatbestow an aggressive phenotype upon NB cells.

The data do not provide evidence that increased expressionof MYCN enhances expression of LDHA. LDHA mRNA was notincreased in the NB of TH-MYCN mice compared with non-tumorous sympathetic ganglia in wild-type mice, in line withother data (41, 42). In our limited number of NB cell lines, nodifference in LDHA transcript and protein levels was foundbetween MYCN-amplified and nonamplified cell lines. Acuteactivation of MYCN in SH-EP MYCN-ER NB cells did notinduce LDHA and MYCN did not cooperate with HIF-1a to

induce LDHA. These data do not contradict the finding of usand others (20) that LDHA mRNA and protein are increased inpatient NB with amplification of MYCN, given that chromo-somal amplification of 2p, where MYCN is located, also ampli-fies additional genes which may induce LDHA. Our dataapparently contrast with reports showing that knockdown ofMYCN in LAN-5 NB cells decreases LDHA mRNA and thatMYCN cooperates with HIF-1a to induce LDHA (20). This maybe explained by species- and cell type-specific differences, andby MYCN and HIF-1a being knocked down rather than acti-vated. Other events yet to be elucidated may drive LDHAexpression while impacting on aggressiveness of NB, either viaLDHA or independent thereof.

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Figure 3.

LDHA is expressed in NB of TH-MYCN miceand in human NB cell lines, independent ofMYCN. A, LDHA mRNA does not increaseduring MYCN-induced transformation ofsuperior cervical ganglion cells to NB. mRNAexpression of ODC1 and LDHA during NBprogression from tumor-prone ganglia at 2weeks to tumors at 6 weeks in TH-MYCNþ/þ

mice (red) is shown and compared withganglia in wild-type mice (blue). Linearregression analysis was performed andP values were determined. �� , P < 0.001;n.s., not significant. B, LDHA and LDHB areexpressed in human NB cell linesindependent of MYCN amplification. MYCN-amplified and nonamplified human NB celllines and two primary NB cultures (U-NB1and U-NB2) were analyzed by qRT-PCR.LDHA mRNA expression is shown relative toHPRT expression. The means of replicates ofindividual cell lines are shown in the upperleft panel, the means of all MYCNnonamplified versus all amplified cell lines inthe upper right panel. Protein levels of LDHAand LDHB were determined byimmunoblotting, with Tubulin as control(bottom). Statistical analysis was performedusing the Student t test. n.s., not significant.C, Forced overexpression of MYCN does notsignificantly increase expression of LDHAand HIF-1a in SH-EP NB cells. SH-EP-MYCN-ER cells were treated either with vehicle (�)or with tamoxifen (þ) for 48 hours. UsingqRT-PCR, the amount of ODC1, LDHA, andHIF-1a mRNA in relation to Actin wasdetermined in duplicates. Results representthree independent experiments, calculatedby the 2�DDCt method. Statistical analysiswas performedwith the unpaired, two-tailedStudent t test. �� , P < 0.01.

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LDHA depletion in SK-N-AS and KELLY NB cells decreases clonogenicity, tumorigenicity, and tumor growth. A, LDHA protein is depleted in CRISPR/Cas9knockout clones. SK-N-AS and KELLY cell clones expanded from single cells after transfection with CRISPR/Cas9 and sgRNA1 or sgRNA3 were probed for LDHA byWestern blot analysis. Actinwas used as loading control.B, LDHAdepletion decreases clonogenicity.Wild-type (wt) and homozygous LDHA knockout (ko) clones ofSK-N-AS cells (wt clones sgRNA1 11 and 12, and sgRNA3 1 and 12; ko clones sgRNA1 4 and 8, and sgRNA3 3, 4, and 6) and of KELLY cells (wt clonessgRNA1 1 and 3, and sgRNA3 8; ko clones sgRNA1 4 and 7, and sgRNA3 2, 5, and 6) were used. Clones were seeded at low density into soft agar (KELLY) oronto plastic (SK-N-AS). Colonies were stained and counted. Shown are the means and SD of three independent experiments with the sample size (n) indicated.Statistical analysiswas performed using the Student t test. �� , P <0.01; � , P <0.05. C, LDHA depletion decreases tumor incidence and increases tumor latency. A totalof 1 � 106 cells of SK-N-AS clones (wt clones sgRNA1 11 and sgRNA3 1; ko clones sgRNA3 3 and 4) and 5 � 105 cells of KELLY clones (wt clones sgRNA1 3 andsgRNA3 8; ko clones sgRNA1 7 and sgRNA3 2) were injected subcutaneously into Rag2�/� cgc�/� mice (n ¼ 10–13 per group). The development of tumorswas determined regularly. Shown is the percentage of tumor-free mice at times after transplantation. Statistical analysis was performed using the log-rank test.D, LDHA depletion decreases tumor growth. Size of the tumors generated in C was measured sequentially using a caliper. Tumor volumes and tumor-doubling times of individual tumors were calculated. Statistical analysis was performed using the Student t test; � , P < 0.05; n.s., not significant.






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LDHA is dispensable for the Warburg effect in SK-N-AS, KELLY, and SK-N-BE(2)C cells. A, LDHA depletion in SK-N-AS and KELLY cells does not abrogateLDH activity and does not significantly inhibit aerobic glycolysis. Wild-type (wt) and homozygous LDHA knockout (ko) clones of SK-N-AS cells (wt clones sgRNA1 11and 12, and sgRNA3 12; ko clones sgRNA1 4, and sgRNA3 3 and 6) and of KELLY cells (wt clones sgRNA1 1 and 3, and sgRNA3 8; ko clones sgRNA1 4 and 7,and sgRNA3 2) were cultured for the times indicated. LDH activity, glucose, and lactate were determined in the medium normalized to cell numbers. Shownare the means and SD of three independent experiments with the sample size (n) indicated. P values were determined using the Student t test. �� , P < 0.001;� , P < 0.05; n.s., not significant. B, SK-N-BE(2)C NB tumors constitutively lack LDHA. SK-N-AS, LAN-5, and SK-N-BE(2)C cell lines were injected subcutaneouslyinto RAG2�/�/ cgc�/� mice. Formalin-fixed and paraffin-embedded tumors were stained for human LDHA. Bars equal 100 mm, in the insets 50 mm. C, IntactWarburg effect in SK-N-BE(2)C NB cells despite complete constitutive lack of LDHA. Glucose, lactate, and ATP per gram of tissue mass were determined byinduced metabolic bioluminescence imaging in cryostat sections of subcutaneous tumors derived from SK-N-AS (n¼ 8), LAN-5 (n¼ 10), and SK-N-BE(2)C (n¼ 6).P values were calculated using the Mann–Whitney test. �� , P < 0.001; � , P < 0.05; n.s., not significant.


Dorneburg et al.




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Combined depletion of LDHA and LDHB in SK-N-AS and KELLY cells does not abrogate LDH activity and aerobic glycolysis while ablating clonogenicity. A, LDHB isexpressed in NB clones depleted of LDHA. Cell lysates from SK-N-AS and KELLY clones were used for Western blot analysis of LDHA and LDHB. Actin was used tocontrol for equal loading. Shown is one representative result of three independent Western blots. B, LDHB is strongly decreased after shRNA induction in wild-typeandLDHAknockout cells,whereas LDHCremains not expressed.Wild-type (wt) andLDHAknockout (ko) clonesof SK-N-AScells andofKELLYcellswere stably transducedwith inducible nonsilencing shRNA (ns) or silencing shRNA against LDHB (sh2 and sh3). shRNA expression was induced by doxycycline treatment for 72 hours. LDHA,LDHB, and LDHC proteins were detected by Western blot analysis, and Actin was used as loading control. C, Combined depletion of LDHA and LDHB does not abrogateLDH activity and does not reduce aerobic glycolysis. Wild-type (wt) and LDHA knockout (ko) clones of SK-N-AS cells and of KELLY cells expressing ns, sh2, or sh3 werecultured. LDH activity, determined in cell lysates, and glucose and lactate in culture supernatants were normalized to cell numbers. Shown are the results of threeindependent experiments.P valueswere determinedusing two-wayANOVA. �� ,P<0.001; �� ,P<0.01; � ,P<0.05; n.s., not significant.D,Combineddepletionof LDHAandLDHB ablates clonogenicity. LDHA wt and ko clones of SK-N-AS and KELLY cells expressing ns, sh2, and sh3 were seeded at low density onto plastic. Colonies werestained and counted. Shown are the results of three independent experiments. Statistical analysis was performed using two-way ANOVA. ��, P < 0.001; � , P < 0.05.






We show that LDHA contributes to aggressiveness of NB cells invitro and in vivowhile being dispensable for theWarburg effect. Byusing CRISPR/Cas9-mediated knockout of LDHA and by analyz-ing several cellular clones verified at the genomic and proteinlevel, complete absence of LDHAwas assured.Given the completelack of LDHA, the decrease of aggressiveness was surprisinglymoderate, if one assumes that LDHA plays a pivotal and non-redundant role in theWarburg effect and that aerobic glycolysis iscrucial forNB cells. It is possible that during the selection of LDHAknockout clones cells may have grown out that depend less onLDHA. In addition, the oncogenic context of LDHA may deter-mine its role in NB. Along this line, lack of LDHA did not decreasedevelopment of lymphoma driven by c-MYC in transgenic mice(43), in contrast to RAS-driven tumors that were susceptible toLDHA ablation (43, 44). In NB cells, the balance betweenMYCN,which belongs to the MYC family of transcription factors, andactivated RAS, present in someNB including the SK-N-AS cell line,may influence their response to LDHA depletion. Further sup-porting the notion that oncogenic context influences the role ofLDHA, outcome after depletion of LDHA in other cancers hasranged from severely diminished tumorigenicity (45, 46) to noeffect (47).

The surprisingly moderate decrease of aggressiveness by LDHAdepletion may also be explained by the LDHB isoform, that wehave shown to be amply expressed in NB, substituting for LDHA.By using inducible shRNA, we achieved near-complete depletionof LDHB. Depletion of LDHB decreased growth of NB cells whilenot impacting on the Warburg effect. The former may have beencaused by loss of LDHB-mediated control of lysosomal functionand thus decreased autophagy, as described to occur specifically incancer cells (32). Combined depletion of LDHB and LDHAablated clonogenicity.

Of note, LDH activity was not ablated despite homozygousknockout or constitutive lack of LDHA, andglucose consumption,lactate production, and generationof ATPweremaintained in vitroand in vivo. This shows that LDHA can be dispensable for theWarburg effect in NB cells, a possibility entertained previously(41, 42, 48).

Intriguingly, combined depletion of LDHA and LDHB did notabrogate LDH activity. This may be explained by the residualexpression of LDHB observed because of incomplete knockdown.LDHC protein, which might have substituted for LDHA andLDHB, was not expressed in the cells, in line with near-absentmRNA expression of LDHC in NB cell lines and patient NB.

Strikingly, concomitant depletion of LDHA and LDHB didnot decrease aerobic glycolysis, possibly because of the residual

LDH activity. An alternative explanation could be that LDHmay be dispensable for aerobic glycolysis in NB cells. Irrespec-tively, it can be concluded that therapeutic inhibition of aerobicglycolysis in NB by targeting LDHwill be challenging. However,inhibition of LDHA and LDHB decreased growth of NB cellsindependent of the Warburg effect. Elucidation of the mechan-isms involved and of the impact on nonmalignant cells iswarranted.

In summary, high expression of LDHA in NB is independentlyassociated with poor patient survival and inhibiting LDHA andLDHB decreases NB growth independent of aerobic glycolysis.Thismay have implications for future risk assessment and therapyof NB patients.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C. Dorneburg, C. BeltingerDevelopment ofmethodology:C. Dorneburg,W.Mueller-Klieser, L. Christner,C. BeltingerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Dorneburg, M. Fischer, T.F.E. Barth, W. Mueller-Klieser, B. Hero, D.R. Carter, K. de Preter, L. Christner, F. Speleman,G.M. Marshall, C. BeltingerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Dorneburg, M. Fischer, T.F.E. Barth, W. Mueller-Klieser, B. Hero, J. Gecht, D.R. Carter, K. de Preter, B. Mayer, L. Christner,F. Speleman, G.M. Marshall, C. BeltingerWriting, review, and/or revision of the manuscript: C. Dorneburg, M. Fischer,T.F.E. Barth, Judith Gecht, K. de Preter, B. Mayer, F. Speleman, G.M. Marshall,K.-M. Debatin, C. BeltingerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Fischer, K.-M. Debatin, C. BeltingerStudy supervision: C. Beltinger

AcknowledgmentsThe authors thankAnneleenBeckers for helpwith the TH-MYCNmouse data,

Ali Gawanbacht for FACS sorting, and Nicole Heymann and Helgard Knaußfor technical assistance. This work was supported in part by a grant from theDeutsche Krebshilfe to C. Beltinger (grant number 70112002).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 28, 2017; revised April 20, 2018; accepted June 12, 2018;published first June 20, 2018.

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2018;24:5772-5783. Published OnlineFirst June 20, 2018.Clin Cancer Res Carmen Dorneburg, Matthias Fischer, Thomas F.E. Barth, et al. Aerobic GlycolysisDepletion Decreases Neuroblastoma Growth Independent of LDHA in Neuroblastoma Is Associated with Poor Outcome and Its

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