-
Metabolism and Chemical Biology
Aberrant FGFR Tyrosine Kinase SignalingEnhances the Warburg
Effect by ReprogrammingLDH Isoform Expression and Activity
inProstate CancerJunchen Liu1,2, Guo Chen1,2, Zezhen Liu1,2,
Shaoyou Liu1,2, Zhiduan Cai1,2,Pan You3, Yuepeng Ke2, Li Lai2, Yun
Huang2, Hongchang Gao4,Liangcai Zhao4, Helene Pelicano5, Peng
Huang5,Wallace L. McKeehan2,Chin-Lee Wu6, Cong Wang4,Weide
Zhong1,7, and Fen Wang2
Abstract
The acquisition of ectopic fibroblast growthfactor receptor 1
(FGFR1) expression is well docu-mented in prostate cancer
progression. How it con-tributes to prostate cancer progression is
not fullyunderstood, although it is known to confer a
growthadvantage andpromote cell survival.Here,we reportthat FGFR1
tyrosine kinase reprograms the energymetabolismof prostate cancer
cells by regulating theexpression of lactate dehydrogenase (LDH)
iso-zymes. FGFR1 increased LDHA stability throughtyrosine
phosphorylation and reduced LDHBexpression by promoting its
promoter methylation,thereby shifting cell metabolism from
oxidativephosphorylation to aerobic glycolysis. LDHAdeple-tion
compromised, whereas LDHB depletionenhanced the tumorigenicity of
prostate cancercells. Furthermore, FGFR1 overexpression andaberrant
LDH isozyme expression were associatedwith short overall survival
and biochemical recur-rence times in patients with prostate
cancer.Our results indicate that ectopic FGFR1 expressionreprograms
the energy metabolism of prostatecancer cells, representing a
hallmark change inprostate cancer progression.
Significance: FGF signaling drives theWarburg effect through
differential regulation of LDHA and LDHB, thereby promotingthe
progression of prostate cancer.
Graphical Abstract:
http://cancerres.aacrjournals.org/content/canres/78/16/4459/F1.large.jpg.
Cancer Res; 78(16); 4459–70.�2018 AACR.
© 2018 American Association for Cancer Research
suppressing LDHB expression in prostate cancer.
– FGFR1 signaling
HSP
G
Glucose
O2
CO2
Pyruvate
LDHBLDHA
Lactate
+ FGFR1 signaling
HSP
G
Glucose
O2
CO2
Pyruvate
LDHBLDHA
Lactate
P P
P P
1Department of Urology, Guangdong Key Laboratory of Clinical
MolecularMedicine and Diagnostics, the Second Affiliated Hospital
of South China Uni-versity of Technology, Guangzhou, China.
2Institute of Biosciences and Tech-nology, College of Medicine,
Texas A&M University, Houston, Texas. 3XianyueHospital, Xiamen,
China. 4Wenzhou Medical University, Wenzhou, China.5Departments of
Translational Molecular Pathology, MD Anderson CancerCenter,
Houston, Texas. 6Departments of
PathologyandUrology,MassachusettsGeneral Hospital and Harvard
Medical School, Boston, Massachusetts. 7Depart-ment of Urology,
Guangzhou Medical University, Guangzhou, China.
Note: Supplementary data for this article are available at
Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
J. Liu, G. Chen, and Z. Liu contributed equally to this
article.
Corresponding Authors: Fen Wang, Institute of Biosciences and
Technol-ogy, Texas A&M Health Science Center, Houston, TX
77030-3303. Phone:713-677-7522; Fax: 713-677-7512; E-mail:
[email protected]; WeideZhong, Department of Urology, Guangdong
Key Laboratory of ClinicalMolecular Medicine and Diagnostics,
Guangzhou First People's Hospital,the Second Affiliated Hospital of
South China University of Technology,Guangzhou 510180, China.
E-mail: [email protected]; and Cong Wang,Wenzhou Medical
University, Wenzhou, Zhejiang, China, E-mail:[email protected]
doi: 10.1158/0008-5472.CAN-17-3226
�2018 American Association for Cancer Research.
CancerResearch
www.aacrjournals.org 4459
on June 22, 2021. © 2018 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
Published OnlineFirst June 11, 2018; DOI:
10.1158/0008-5472.CAN-17-3226
http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-17-3226&domain=pdf&date_stamp=2018-7-28http://cancerres.aacrjournals.org/content/canres/78/16/4459/F1.large.jpghttp://cancerres.aacrjournals.org/
-
IntroductionMetabolic reprograming from oxidative
phosphorylation to
aerobic glycolysis is a common event in cancer
progression.Although glycolysis is less efficient than oxidative
phosphoryla-tion for providing energy with respect to the number of
ATP perglucose, it meets the demand of rapidly growing cancer cells
forbuilding blocks. In addition, increased glycolysis results in
glu-cose deprivation and lactate accumulation in the tumor
micro-environment, which suppresses lymphocyte infiltration and
com-promises anti-immunotherapies (1, 2). In line with those
effects,the glycolytic phenotype is associated with prostate cancer
pro-gression and aggressiveness (3–10). An understanding of
howtumor cells reprogram their metabolism from oxidative
phos-phorylation to aerobic glycolysismay provide a novel approach
toselectively inhibit aerobic glycolysis in tumor cells.
The prostate consists of epithelial and stromal
compartments,which maintain active two-way communication through
para-crine mechanisms, including fibroblast growth factor (FGF)
sig-naling in which FGF and FGF receptor (FGFR) are
partitionedbetween the two compartments (11). That precisely
balancedcommunication is critical for preserving the tissue
homeostasisand function of the prostate. The FGF family consists of
18 ligandsthat exert their regulatory signals by activating the
FGFR tyrosinekinases encoded by 4 homologous genes. FGF and FGFR
areexpressed throughout the body in a pattern that is
spatiotempo-rally and cell-type specific, controlling embryonic
developmentand maintaining adult tissue homeostasis and
function.
There is extensive evidence that ectopic activation of
theFGF/FGFR signaling axis is associated with prostate cancer
devel-opment and progression (11). The acquisition of ectopic
FGFR1expression stands out as the most remarkable change among
theFGFR isotypes in prostate cancer. The forced expressions of
FGFand FGFR have been shown to induce prostate lesions in
mousemodels. On the other hand, the ablation of Fgfr1 or Frs2a,
whichencodes FGFR-substrate 2a (FRS2a), an adaptor protein
neededfor FGFR kinases to activate ERK and PI3K/AKT pathways,
signif-icantly reduced prostate cancer development and progression
inmice (12, 13).
FGF signaling promotes aerobic glycolysis by increasing
hexo-kinase 2 (HK2) expression and the tyrosine phosphorylation
ofmultiple enzymes involved in aerobic glycolysis (14). Whetherand
how ectopic FGF signaling contributes to prostate cancerprogression
by promoting aerobic glycolysis, remains to be deter-mined. The
last step of glycolysis is the reduction of pyruvate tolactate, a
reversible conversion catalyzedby lactate dehydrogenase(LDH). LDH
is a tetrameric enzyme composed of two types ofsubunits, LDHA and
LDHB. The combination of the two subunitsyields five isozymes,
which catalyze the conversion betweenpyruvate and lactate. LDH1 is
composed of four LDHB subunitsand favors the conversion from
lactate to pyruvate, allowingoxidation along the pathway of the
tricarboxylic acid cycle. LDH5is composed of four LDHA subunits and
favors the conversionfrom pyruvate to lactate, allowing the
glycolytic pathway to becompleted at the formation of lactate (15).
Interestingly, hypoxiainduces LDHA, while inhibits LDHB, expression
(16, 17). Emerg-ing evidence shows that LDHA is required for the
survival andproliferation of cancer cells. Although still a matter
of debate,current data seem to indicate an association between
reducedlevels of LDHB and increased malignancy in prostate cancer
andother cancers (18–27).
LDHA is tyrosine phosphorylated in cancer cells (28). FGFR1has
been reported to phosphorylate LDHA at multiple tyrosineresidues,
which enhances tetramer formation andNADHbindingand thus the
enzymatic activity of LDHA (29). In this study, wedemonstrate that
FGFR1 signaling promotes aerobic glycolysis bystabilizing LDHA
through tyrosine phosphorylation and down-regulating LDHB
expression through the induction of hyper-methylation in the Ldhb
promoter. We found that LDHA ablationcompromised, whereas LDHB
ablation enhanced, the tumorige-nicity ofDU145 cells. Furthermore,
high levels of phosphorylatedLDHA and low levels of LDHB in human
prostate cancer tissueswere associated with short biochemical
recurrence and survivaltimes in patients with prostate cancer.
Together, our resultssuggest that ectopically expressed FGFR1 in
prostate cancerinduces metabolic changes by increasing LDHA levels
and low-ering LDHB levels, which promotes prostate cancer growth
andprogression.
Materials and MethodsAnimals
Mice were housed under the Program of Animal Resources ofthe
Institute of Biosciences and Technology in accordance withthe
principles and procedures of the Guide for the Care andUse of
Laboratory Animals. All experimental procedures wereapproved by the
Institutional Animal Care and Use Committee.Mice were bred and
genotyped as described previously(12, 30, 31). For xenograft
studies, 2 � 106 DU145 cells weremixed with Matrigel (BD
Biosciences) at a 1:1 ratio and subcu-taneously injected into the
flanks of nude mice (6-week old).The size of xenograft was measured
with a caliper and calculatedasV¼ 0.52� length�width2. The
xenografts were harvested afterthe animals were euthanized via CO2
suffocation.
HistologyProstate tissues and xenografts were fixed, embedded,
sec-
tioned as described (32). For immunostaining, the antigens
wereretrieved by boiling in citrate buffer (10 mmol/L, pH 8.0) for
20minutes. Rabbit antiphosphorylated LDHA (pLDHA, 1:200)and
anti-LDHA (1:200) were purchased from Cell SignalingTechnology.
Mouse anti-LDHB (1:200), rabbit anti-CD31(1:200) and anti-Ki67
(1:200), and rat anti-F4/80 (1:200) werepurchased from Abcam. A
Fluoremetric TUNEL Assay Kit waspurchased from Premega Co. The
ExtraAvidin Peroxidase System(Sigma Aldrich) and
fluorescence-conjugated secondary antibo-dies (Invitrogen) were
used to visualize specifically bound anti-bodies. For
immunofluorescence staining, the nuclei were coun-terstained with
To-Pro 3 before being observed under a confocalmicroscope (Zeiss
LSM 510).
The Massachusetts General Hospital (Boston, MA) prostatecancer
tissue microarray (TMA) was used to assess the expres-sion of LDHA,
LDHB, and Fgfr1 as well as the level of phos-phorylated LDHA in
human prostate cancer samples asdescribed previously (33).
Immunostaining of prostate cancercells and that of stromal cells
was evaluated separately. Thepercentage of positive cells was
calculated and categorized asfollows: 0, 0%; 1, 1%–10%; 2, 11%–50%;
3, 50%–75%; and 4,75%–100%. The staining intensity was visually
scored anddefined as follows: 0, negative; 1, weak; 2, moderate;
and 3,strong. Final immunoreactivity scores (IRS) were calculated
foreach case by multiplying the percentage and intensity
scores.
Liu et al.
Cancer Res; 78(16) August 15, 2018 Cancer Research4460
on June 22, 2021. © 2018 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
Published OnlineFirst June 11, 2018; DOI:
10.1158/0008-5472.CAN-17-3226
http://cancerres.aacrjournals.org/
-
Western blottingCells or xenografts were homogenized in RIPA
buffer as
described previously (32). Samples containing 30 mg
proteinwereseparated by SDS-PAGE and blotted onto polyvinylidene
difluor-ide membranes. The antiphosphorylated ERK1/2 (1:1,000),
anti-phosphorylated AKT (1:1,000), anti-ERK1/2 (1:1,000),
anti-AKT(1:1,000), antiphosphorylated FRS2a (1:1,000), and
anti-HA(1:1,000) antibodies were purchased from Santa Cruz
Biotech-nology. Anti-pLDHA (1:1,000), anti-LDHB (1:1,000), and
anti-LDHA 1:1,000) antibodies and the Glycolysis Antibody
SamplerKit containing antibodies against HK1, PFKP, PFKP2,
PFKP3,aldolase, PGAM, PKM1/2, and pyruvate dehydrogenase
werepurchased from Cell Signaling Technology. The
antiphosphotyr-osine 4G10 (1:1,000), anti-TET1 (1:1,000), and
anti-Flag(1:1,000) antibodies were purchased from Millipore Sigma.
Thespecifically bound antibodies were visualized using the
ECL-Pluschemiluminescent reagents. The films were scanned with a
den-sitometer for quantitation.
RNA expressionTotal RNA was isolated from cells and tissues
using the Ribo-
pure RNA isolation reagent (Ambion), reverse transcripted
withSuperScript III (Life Technologies) and random primers,
andanalyzed with real-time PCR using the Fast SYBR Green MasterMix
(Life Technologies) as described. The results were expressed asthe
mean� SD as described (32). The primer sequences are listedin
Supplementary Table S1.
Gene ablationThe lentivirus-based CRISPR-Cas9 system was used to
ablate
the Ldha, Ldhb, and Fgfr1 alleles in DU145 cells. The sequences
ofthe sgRNAs are shown in Supplementary Table S1. Two days
afterinfection with the lentivirus, the recombinant cells were
selectedvia growth in a medium containing 2 mg/mL puromycin.
Site-directed mutagenesisThe QuikChange Lightning Multi
Site-Directed Mutagenesis
Kit (Agilent Technologies) was used to generate
LDHA-mutantcDNAs. The primer sequences are listed in
SupplementaryTable S1.
NMR analysesCells (2 � 107) were suspended in 450 mL
methanol/chloro-
form (2:1) and lysed by ultrasound. The lysates were mixed
with450 mL chloroform/H2O. The supernatants were lyophilized
andthen resuspended in 500-mL D2O containing 0.25 mmol/L sodi-um
trimethylsily 1 propionate-d4. All NMR spectra were recordedon a
Bruker AVANCE III 600 MHz NMR spectrometer.
Methylated DNA immunoprecipitationCells were isolated with the
Qiagen Kit and ultrasound shear-
ed into smaller fragments (200–600 bp). The sheared gDNAwas
denatured by incubation in 1 mol/L NaOH/25 mmol/LEDTA at 95�C for
12 minutes, and then immunoprecipitatedwith anti-5-methylated
cytosine (5mC) antibodies. Thebound 5mC-containing DNA was then
purified and used tomake the sequencing library with the NEB
(E6240S) LibraryPrep Kit, which was subsequently sequenced by the
AgrelifeGenomics and Bioinformatics Service, Texas A&M
University(College Station, TX).
Sodium bisulfite DNA sequenceGenomic DNA was isolated using the
Qiangen Genomic DNA
Kit, followed by bisulfite conversion using the EpiJET
BisulfiteConversion Kit (Thermo Scientific). The bisulfite-specific
primersfor the PCR amplification were listed in Supplementary Table
S1.The PCR products were cloned into pDrive Cloning
Vectors(Qiangen) and sequenced.
Protein stability assayStable MEF transfectants expressing
HA-tagged LDHA were
treated with cycloheximide for the indicated times. The
abun-dance of LDHA was examined by Western blot analysis.
Therelative level of endogenous or HA-tagged LDHAwas
quantitatedusing ImageJ. The GraphPad software was used to compare
theslopes of each curve.
Statistical analysisStatistical analysiswas performedusing the
two-tailed t test. For
protein stability assay, the GraphPad software was used to
com-pare two curves. P < 0.05 is considered statistical
significant. Errorbars indicate SDs.
ResultsAblation of the FGF signaling axis reduces glycolysis
andincreases oxygen consumption in MEF
We first determined whether FGF signaling regulated
cellmetabolism in mouse embryonic fibroblasts (MEF), whichexpressed
FGFR1, FGFR2, and FRS2a. We generated MEFs thatwere devoid of
Fgfr1, Fgfr2, and Frs2a by infecting MEFs bearingfloxed Fgfr1,
Fgfr2, and Frs2a alleles with adenovirus carrying theCre-GFP coding
sequence. The cells, designated MEFDF, did notexpress Fgfr1, Fgfr2,
and Frs2amRNA (Fig. 1A). Interestingly, real-time RT-PCR also
revealed that at the mRNA level, MEFDF
increased Ldhb, but not Ldha expression. However, Western
blotanalyses revealed that at the protein level, comparedwith
parentalMEFs, the MEFDF cells had reduced LDHA expression
andincreased LDHB expression (Fig. 1B). Those results suggest
thatFGF signaling promotes LDHA expression at the protein level
andsuppresses LDHB expression at the mRNA level. Western
blotanalysis also showed that FGF2 failed to induce
phosphorylationof FRS2a, ERK, and AKT, indicating successful
abrogation of FGFsignaling in the cells.
LDHA and LDHB favor opposite direction of the conversa-tion
between pyruvate and lactate (15). To determine whetherabrogation
of the FGF signaling axis in MEFs changes the cellmetabolism, we
compared the lactate production and oxygenconsumption in MEFs and
MEFDF cells (Fig. 1C). The abro-gation of FGF signaling reduced
lactate production andincreased the oxygen consumption rate. In
contrast, overexpres-sion of LDHA in MEFDF cells significantly
increased lactateproduction (Supplementary Fig. S1). In addition,
NMR anal-yses revealed that the abrogation of FGF signaling reduced
theconcentrations of isoleucine, valine, lactate, and acetateand
increased the concentrations of glutamate and succinate(Fig. 1D).
The reduced isoleucine and valine levels in MEFDF
cells suggest that the synthesis of branched chain amino
acids(BCAA) was compromised. The dysregulation of BCAA synthe-sis
in MEFs lacking FGF signaling is in line with evidence thatBCAAs
promote glucose uptake (34). Those results demonstratea shift in
energy metabolism from aerobic glycolysis to
FGF Signaling in Cell Energy Metabolism
www.aacrjournals.org Cancer Res; 78(16) August 15, 2018 4461
on June 22, 2021. © 2018 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
Published OnlineFirst June 11, 2018; DOI:
10.1158/0008-5472.CAN-17-3226
http://cancerres.aacrjournals.org/
-
oxidative phosphorylation in MEFDF cells, suggesting that
ener-gy metabolism in MEFs is regulated by FGF signaling.
FGFR1 enhances the stability of LDHA via
tyrosinephosphorylation
To determine the role of FGFR1 in the regulation of LDHA atthe
protein level, we treated the MEFs and MEFDF cells
withcycloheximine to block protein synthesis. Western blot
analysesrevealed that the abundance of LDHA declined faster in
MEFDF
cells than in the parental MEFs (Fig. 2A). Those results
indicatethat the half-life of LDHA is shorter in MEFDF cells than
in theparental MEFs, suggesting that FGF signaling enhances
thestability of LDHA.
LDHA consists of four tyrosine phosphorylation sites(Fig. 2B).
In line with a previous report that FGFR1 directlyphosphorylates
LDHA (29), Western blot analyses with theantiphosphotyrosine
antibody 4G10 showed that LDHA wastyrosine phosphorylated (Fig.
2C). To identify which tyrosinephosphorylation sites were involved
in the regulation of LDHAstability, we employed site-directed
mutagenesis to generateLDHAmutants with individual, double (2F), or
quadruple (4F)mutations. The individual mutations at each of the
four tyro-sine-phosphorylation sites did not affect the
phosporylationLDHA. Note that weak expression of FGFR1 in the Y83F
groupmight account for the relatively low phosphorylation of
Y83F.Only the 4F mutant failed to be phosphorylated by FGFR1.
Theother mutants displayed only partially reduced phosphoryla-tion,
comfirming the previous report that all four tyrosineresidues are
phosphorylated by FGFR1.
Western blot analyses revealed that the individual mutationsat
each of the four tyrosine phosphorylation sites and the two2F
mutants did not affect the stablility of LDHA (P > 0.05).
However, the 4F mutant showed a statistically
significantreduction in stabilitity (Fig. 2D). Together, those
results indi-cate that phosphorylation of the four tyrosine
residues pro-motes the stability of LDHA.
FGFR1 suppresses LDHB transcription by promoting DNAmethylation
in the LDHB promoter
The Ldhb promoter is heavily methylated in prostate cancer,which
inhibits the transcription of LDHB (22). To determinewhether FGF
signaling suppresses LDHB expression via pro-moter methylation, we
employed methylated DNA immuno-precipitation to pull down the
methylated DNA for high-throughput sequencing (Fig. 3A). The
results showed that theCpG islands in the Ldhb promoter were less
methylated inMEFDF cells than in the parental MEFs. Further
bisulfitesequencing of the Ldhb promoter area confirmed that
DNAdemethylation was reduced in the MEFDF cells (Fig. 3B).
Con-sistent with the data that ablation of FGF signaling did
notaffect LDHA mRNA expression, no differences were observed inldha
promoter methylation between MEF and MEFDF cells(Supplementary Fig.
S2).
Because DNA demethylation is catalyzed by the three TETenzymes
(TET1–3), we compared the expression levels ofTET1–3 in MEFDF cells
and parental MEFs. We found that theexpression of Tet1 at both mRNA
and protein levels wassignificantly increased in the MEFDF cells
(Fig. 3C and D),suggesting that FGF signaling suppressed Tet1
expression. Con-sistantly, Ldhb, but not ldha, expression was
higher in MEFsbearing Tet1null alleles than in parental MEFs (Fig.
3E), furtherindicating that the expression of LDHB was reduced by
DNAmethylation.
Figure 1.
Ablation of FGF signaling suppresses aerobic glycolysis and
promotes oxidative phosphorylation in MEFs.A, Real-time RT-PCR
analyses of the indicated mRNAs. B,Western blot analysis of the
indicated proteins. C, Relative oxygen consumption and lactate
production. D, NMR analyses demonstrated metabolite changesin MEFDF
cells. Each column represents an individual sample. WT, wild-type;
Ctrl, control; DF, MEFDF. � , P < 0.05.
Liu et al.
Cancer Res; 78(16) August 15, 2018 Cancer Research4462
on June 22, 2021. © 2018 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
Published OnlineFirst June 11, 2018; DOI:
10.1158/0008-5472.CAN-17-3226
http://cancerres.aacrjournals.org/
-
Ablation of the FGF signaling axis reprograms cell metabolismin
human prostate cancer cells
DU145 cells highly expressed Fgfr1 (Fig. 4A). To
investigatewhether ectopic FGF signaling contributes to metabolic
repro-
gramming in prostate cancer, we used CRISPR/Cas9 geneediting to
ablate the Fgfr1 alleles in DU145 cells that expressedhigh levels
FGFR1. Although it did not affect cell growth inthe medium with 10%
FBS, ablation of Fgfr1 compromised cell
Figure 2.
Tyrosine-phosphorylation suppresses thedegradation of LDHA. A,
The cells were treated withcycloheximide (CHX) for the indicated
times.Endogenous LDHA proteins were determined byWestern blot
analysis. The intensity of LDHA relativeto that of b-actin is shown
on the right. B, Thestructural domains of LDHA. Green dots,
tyrosinephosphorylation sites. C, HA-tagged LDHA wasexpressed in
239T cells with FGFR1. PhosphorylatedLDHA was detected with
antiphosphotyrosineantibody 4G10. D, The cells were treated
withcycloheximide for the indicated times. The levels ofLDHA were
determined by Western blot analysis.The intensity of LDHA relative
to that of b-actin isshown on the right. T, tetramer formation
domain;NADP, NAD/NADP binding domain; C, C-terminaldomain; 2Fa,
Y10F/Y83F mutation; 2Fb, Y172/Y239mutation; 4F, Y10F/Y83F/Y172/Y239
mutation; IP,immunoprecipitation; IB, immunoblot. � , P <
0.01.
Figure 3.
Ablation of FGF signaling reduces DNA methylation in the Ldhb
promoter. A, Methylated DNA was immunoprecipitated and subjected to
high-throughputsequencing. The level of methylation at CpG islands
in the Ldhb promoter region is shown. B, Bisulfite DNA sequencing
of the Ldhb promoter area inMEFDF cells. C and D, Real-time RT-PCR
and Western blot analyses showing increased Tet1 in MEFDF cells at
the mRNA and protein levels. E, Real-time RT-PCRanalyses of ldha
and Ldhb in Tet1 null MEF (DTet1). � , P < 0.05.
FGF Signaling in Cell Energy Metabolism
www.aacrjournals.org Cancer Res; 78(16) August 15, 2018 4463
on June 22, 2021. © 2018 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
Published OnlineFirst June 11, 2018; DOI:
10.1158/0008-5472.CAN-17-3226
http://cancerres.aacrjournals.org/
-
growth in the medium with 1% serum (Supplementary Fig. S3).In
line with the data from MEFs, the expression of Ldha mRNAwas not
affected in the Fgfr1null DU145 (DU145DR1) cells,whereas that of
Ldhb mRNA was increased (Fig. 4B). We thendetermined the expression
profiles of the LDH isozymes in theparental and DU145DR1 cells by
Western blot analysis (Fig. 4C).The ablation of Fgfr1 reduced LDHA
expression and increasedLDHB expression at the protein level.
Interestingly, expressionof FGFR1 protein was increased by FGF2
treatment. However,the underlying mechanism remains to be
determined. Togetherwith Fig. 4B, the results further indicate that
FGFR1 regulatesLDHA at either the translational or
posttranslational level andLDHB at the transcriptional level.
Consistent with the reportthat FGFR1 phosphorylates LDHA, Western
blot analysesrevealed that the phosphorylation of LDHA at the Y10
residuewas reduced in the DU145DR1 cells. Consistently, the
ablationof Fgfr1 reduced glucose uptake and lactate production
andincreased O2 consumption (Fig. 4D). Similarly, treating
DU145cells with FGFR inhibitor AZD4547 also suppressed
glycolysis(Supplementary Fig. S4). Together, the data suggest that
theablation of Fgfr1 in prostate cancer cells reprograms the
cellenergy metabolism from oxidative phosphorylation to
aerobicglycolysis.
LDHA ablation reduces, whereas LDHB ablation enhances,
thetumorigenesis of DU145 cells
To investigate the functions of the LDH isoforms in the
tumor-igenic activity of prostate cancer cells, we employed
CRISPR/Cas9to delete the Ldha or Ldhb alleles in DU145 cells,
designatedDU145DLdha andDU145DLdhb, respectively. Western blot
analysesof the xenografts revealed that the ablation of Ldha
increasedPGAM levels, while the ablationof Ldhb increasedHK1, PFK3,
andaldolase levels (Fig. 5A). Those results indicate that LDHA
deple-
tion compromised, whereas LDHB depletion enhanced, glycoly-sis
inDU145 cells. Althoughnodifferencewas observed in growthrates
(Fig. 5B), when grafted subcutaneously into the flanks ofnudemice,
DU145DLdha cells generated smaller tumors comparedwith parental
DU145 cells (Fig. 5C). In contrast, grafts ofDU145DLdhb cells
generated larger tumors than those of parentalDU145 cells.
Consistantly, similar results were derived from PC3cells
(Supplementary Fig. S5).
LDHA deletion and LDHB deletion both stimulated com-pensatory
upregulation of glycolytic-related proteins but hadopposite effects
on tumor growth, suggesting that there mightbe unidentified
proteins that mediate LDH-regulated tumorgrowth. Noticebly,
ablation of Ldha increased expression ofLDHB and vice versa.
Whether these changes are because offeedback regulation to
compensate the loss of LDH remains tobe investigated.
Although there were no significant differences in
tissuehistology among the parental DU145, DU145DLdha, andDU145DLdhb
xenografts, the DU145DLdha xenografts had less,and the DU145Dldhb
xenografts had more, Ki67þ cells than theparental DU145 xenografts,
indicating that the ablation of Ldhareduced, and the ablation of
Ldhb increased, the number ofproliferating cells in the xenografts
(Fig. 6A and B). Further-more, there were more apoptotic cells in
the DU145DLdha
xenografts than in the parental DU145 xenografts, whereas
theopposite relation was observed between the DU145DLdhb
xeno-grafts and the parental DU145 xenografts (Fig. 6C). To
inves-tigate the impact of Ldha or Ldhb ablation on the
tumormicroenvironment, we stained DU145DLdha, DU145DLdhb,
andparental DU145 xenografts with anti-CD31 antibodies
andanti-F4/80 antibodies to examine the densities of
microvesselsand macrophages, respectively. There were fewer CD31þ
andF4/80þ cells in the DU145DLdha xenografts than in the
parental
Figure 4.
Ablation of FGF signaling suppresses aerobic glycolysis and
promotes oxidative phosphorylation in DU145 cells. A and B,
Real-time RT-PCR analyses ofFGFR and LDH isoform expression. C,
Western blot analysis of LDHA and LDHB expression. D, Comparison of
glucose uptake, oxygen consumption,and lactate and ATP production.
Ctrl, control; DR1, DU145DR1; pLDHA, phosphorylated LDHA; pERK,
phosphorylated ERK1/2; pAKT, phosphorylated AKT;b-actin was used as
a loading control. � , P < 0.05.
Liu et al.
Cancer Res; 78(16) August 15, 2018 Cancer Research4464
on June 22, 2021. © 2018 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
Published OnlineFirst June 11, 2018; DOI:
10.1158/0008-5472.CAN-17-3226
http://cancerres.aacrjournals.org/
-
DU145 xenografts. In contrast, there were more CD31þ and F4/80þ
cells in the DU145DLdhb xenografts than in the parentalDU145
xenografts. Those results demonstrated that Ldha abla-tion
compromised, whereas Ldhb ablation stimulated, angio-genesis and
macrophage infiltration and thus the tumorigenicactivity of DU145
cells.
Hyperphosphorylation of LDHA and reduced LDHBexpression levels
in human prostate cancer
We performed IHC staining to determine the expression pat-terns
of LDHA and LDHB in a human prostate TMA thatcomprised 225 prostate
cancer samples and 27 benign prostatesamples (33). The results
suggested that LDHA levels were higherin prostate cancer tissues
than in adjacent prostate tissues(Fig. 7A). FGFR1 can phosphorylate
human LDHA at multipletyrosine residues (29). Compared with
noncancerous tissues,prostate cancer tissues had higher levels of
Y10-phosphorylatedLDHA (pLDHA) and lower levels of LDHB (Fig.
7B).
The samples in the TMA were annotated with detailedinformation
based on a 15-year follow-up of the patients,including PSA
recurrence, Gleason Scores, pathologic stages,patients' age, and
survival time. Therefore, we separated thesamples into two groups
based on the median level. Higherthan the median is defined as High
and lower than the medianis defined as Low. As shown in Fig. 7C and
D, the clinicaloutcomes of the patients with high pLDHA levels (N ¼
153)were worse than those of the patients with low pLDHA levels
(N ¼ 48), while those of patients with low LDHB levels(N ¼ 126)
were worse than those of patients with high LDHBlevels (N ¼ 96);
the patients with high pLDHA levels andlow LDHB levels (N ¼ 13)
clearly had shorter biochemicalrecurrence-free times than those
with low pLDHA levels andhigh LDHB levels (N ¼ 86). Those results
suggest that pLDHAand LDHB expression have the potential to serve
as bio-markers for prostate cancer prognosis.
Fgfr1 is overexpressed in about 40% of human prostatecancers
(35, 36). To determine whether the expression level ofFgfr1
correlates with the abundance of pLDHA, we used in
situhybridization to assess the expression levels of Fgfr1 in the
TMA.Fgfr1 expressed was higher in prostate cancer than in
benigntissues (Fig. 7D). The expression was associated with
shortPSA-free survival time (Fig. 7E). Furthermore, the Fgfr1
expres-sion level was positively associated with the level of
pLDHA(Fig. 7F). Together, the data imply that ectopically
expressedFgfr1 in prostate cancer deregulates the expression of
LDHisozymes and thus changes the glycolysis and metabolism ofthe
cells (Fig. 7G).
DiscussionThere is extensive evidence that ectopic FGF/FGFR1
signaling
is a contributing factor in prostate cancer development
andprogression (11, 36–41), that FGF-mediated glycolysis playsa
pivotal role in development (14, 42), and that FGFR1 directly
Figure 5.
LDHA ablation reduces, and LDHB ablationenhances, the
tumorigenicity of DU145 cells. A,Western blot analysis of the
expression of enzymesrelated to aerobic glycolysis. B, The
indicatedDU145 cells (2 � 104) were plated on 6-cm dishes.Cell
numbers were counted every other day.C, Xenografts derived from the
indicated controlDU145 cells. Note that the DLdha and DLdhb
tumorswere harvested at different days, because theDLdhb tumors
reached the limit of tumor burdenearlier than the DLdha tumors. The
averagexenograft weight was calculated from all
individualxenografts and is presented on the right.
DLDHA,DU145DLdha; DLDHB, DU145DLdhb; Ctrl, control DU145cells; HK1,
hexokinase 1; PD, pyruvatedehydrogenase. � , P < 0.05.
FGF Signaling in Cell Energy Metabolism
www.aacrjournals.org Cancer Res; 78(16) August 15, 2018 4465
on June 22, 2021. © 2018 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
Published OnlineFirst June 11, 2018; DOI:
10.1158/0008-5472.CAN-17-3226
http://cancerres.aacrjournals.org/
-
phosphorylates LDHA and enhances its enzymatic activity(29).
Herein, we showed that FGFR1 signaling promotes aer-obic glycolysis
by upregulating LDHA at the protein level and
downregulating LDHB at the transcriptional level. The ablationof
LDHA compromised, whereas that of LDHB enhanced, thetumorigenic
activity of DU145 prostate cancer cells.
Figure 6.
Differential impacts of LDHA and LDHB on cell survival,
angiogenesis, and inflammation in prostate cancer xenografts. A and
B, Hematoxylin and eosin (H&E)and IHC staining. The numbers of
Ki67þ cells per viewing area were calculated from 20 viewing areas
per tumor from six pairs of tumors and are presented asthe mean �
SD on the right. C, Tissue sections of the xenografts were
immunostained with TUNEL, anti-CD31, or anti-F4/80 antibodies as
indicated. Thenumbers of positively stained cells per viewing area
were calculated from 25 viewing areas from six pairs of tumors and
are presented as the mean � SD.
Liu et al.
Cancer Res; 78(16) August 15, 2018 Cancer Research4466
on June 22, 2021. © 2018 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
Published OnlineFirst June 11, 2018; DOI:
10.1158/0008-5472.CAN-17-3226
http://cancerres.aacrjournals.org/
-
Furthermore, high levels of phosphorylated LDHA and lowlevels of
LDHB in human prostate cancer tissues were associ-ated with short
biochemical-recurrence and survival times ofpatients. Our results
suggest that ectopic FGFR1 signalingcontributes to prostate cancer
progression by reprograming cellenergy metabolism and those high
levels of phosphorylatedLDHA and high expression levels of LDHB are
potential bio-markers for prostate cancer diagnosis and
prognosis.
LDHA has four tyrosine phosphorylation sites. Phosphory-lation
at tyrosine 10 enhances the formation of tetramers andincreases
enzymatic activity, while phosphorylation at tyrosine83 enhances
the binding of NADH (29). In addition, highlevels of FGFR1
expression are associated with high levels ofphosphorylated LDHA
(43). Tyrosine 10 only exists in humanLDHA, suggesting that Y10
phosphorylation is not the onlyway in which FGFR1 activates LDHA.
We showed that
Figure 7.
High pLDHA and low LDHB expression predicts poor prognosis in
patients with prostate cancer. A, Representative images of
immunochemicalstaining of pLDHA and LDHB in the MGH prostate cancer
TMA. B, Statistical analyses of the expression of pLDHA and LDHB in
prostate cancer andbenign prostate. C, PSA failure-free survival
time in patients with low versus high phosphorylated LDHA a LDHB
expression. D, Statisticalanalyses of FGFR1 in benign and cancer
tissues. E, PSA failure-free survival time in patients with low-
versus high-FGFR1 expression. F, Pearsoncorrelation of Fgfr1 and
pLDHA in the prostate cancer TMA. G, Model of ectopic FGFR1
signaling in reprograming cell metabolism and
promotingtumorigenesis.
FGF Signaling in Cell Energy Metabolism
www.aacrjournals.org Cancer Res; 78(16) August 15, 2018 4467
on June 22, 2021. © 2018 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
Published OnlineFirst June 11, 2018; DOI:
10.1158/0008-5472.CAN-17-3226
http://cancerres.aacrjournals.org/
-
phosphorylation of the four tyrosine phosphorylation sites
byFGFR1 extended the half-life of LDHA. Ablation of the
FGFsignaling axis significantly reduced the half-life of LDHA;
thesubstitution of phenylalanine at the four tyrosine residues
inLDHA had the same effect (Fig. 2). Together, the results
suggestthat tyrosine phosphorylation not only increases the
enzymaticactivity of LDHA but also enhances the stability of LDHA.
Moreresearch is needed to determine how tyrosine
phosphorylationaffects the half-life of LDHA.
It has been reported that LDHB expression is silenced
byhypermethylation of the LDHB promoter in human prostatecancer
(23). We found that Fgfr1 ablation reduced the meth-ylation of CpG
islands in the Ldhb promoter (Fig. 3A and B).The ablation of FGF
signaling in MEFs increased the expressionof Tet1, which catalyzes
the conversion of methylated guani-dine to hydroxyl-methylated
guanidine, the first step of DNAdemethylation. Moreover, ablation
of Tet1 increased LDHBexpression although ablation of LDHA or LDHB
did not affectTET1 expression (Supplementary Fig. S6). Thus, our
resultsdemonstrate that FGF signaling suppresses LDHB expressionby
promoting Tet1 expression.
Because LDHA promotes aerobic glycolysis while LDHBfacilitates
oxidative phosphorylation, LDHA may enhancetumor progression by
fueling aerobic glycolysis and LDHBexerts opposite effects in
glycolytic prostate cancer cells. Ourresults in Fig. 4 revealed
that LDHA and LDHB have oppositeeffects on prostate cancer growth
further support this hypoth-esis. Hence, it is essential to develop
new strategies to specif-ically inhibit LDHA without compromising
LDHB activity.FGFR1 selectively phosphorylates LDHA on multiple
tyrosineresidues, which stabilizes LDHA, and concurrently
enhancesthe expression of LDHB. Yet, other signaling pathways may
alsoregulate cell metabolism, which may explain why ablation ofFGF
signaling did not fully convert LDH expression from LDHAto LDHB. It
has been reported that LDHA is degraded viachaperone-mediated
autophagy (44). However, no differencein LDHA degradation was
observed in DU145 and DU145DR1
cells with or without treating with protease inhibitor
leupeptinor autophagy inhibitor bafilomycin (Supplementary Fig.
S7),suggesting that FGF signaling protected LDHA degradation
viaother mechanism. Although both lactate production and glu-cose
consumption are reduced in DU145DR1 cells (Fig. 4), thegrowth
curves of DU145 and DU145DR1 were not significantlydifferent in
glucose-free medium with or without lactate sup-plement, suggesting
that deletion of FGFR1 did not confer thecells ability to use
lactate as a main energy source. Furtherefforts are warranted to
fully understand how cell metabolismis regulated.
Our data are consistent with reports that loss of LDHBcorrelated
with malignancy (20, 26, 45). However, it is dif-ferent from the
report that LDHB expression is associated withpoor survival in
patients with uterine cancer (27, 46). Oneexplanation is that in
the cancer, such as uterine cancer, whichlargely rely on the TCA
cycle to fuel cellular activities, can beinhibited by LDHB
depletion. In other cancers, such as met-astatic prostate cancer,
however, rely on aerobic glycolysis, andtherefore, is benefited
from LDHB depletion. Therefore, morestudies are needed to
understand the role of LDHB in varioustypes of malignancies. Our
data show that although thedeletion of FGFRs led to an increase in
oxygen consumption,it still reduced ATP production. These results
suggest that the
upregulation of oxidative phosphorylation is not sufficientto
make up the energy loss because of FGFR ablation, andthat prostate
cancer cells derive ATP primarily from aerobicglycolysis.
Although FRS2a is required for the FGF kinase to activatethe ERK
and PI3K/AKT pathways, we found that deletion ofFrs2a alleles did
not affect LDHA and LDHB expressions aswell as lactate production
in MEFs (Supplementary Fig. S8).This is consistent with the fact
that FGFR1 directly phos-phorylates LDHA. The mechanism by which
FGF sig-naling regulates LDHB promoter methylation remains to
bedetermined.
In summary, we demonstrated that FGF signaling promotesaerobic
glycolysis and that ectopic FGF signaling in prostatecancer
reprograms cell metabolism, suppressing aerobic glycoly-sis and
promoting oxidative phosphorylation. LDHA deletionsuppressed,
whereas LDHB deletion promoted, the tumorigenicactivity of prostate
cancer cells. Furthermore, the LDHA over-expression and LDHB
downregulation correlated with short bio-chemical recurrence and
survival times in patients with prostatecancer. Our results shed
new light on how the manipulation ofectopic FGF signaling may serve
as a strategy for prostate cancertreatment.
Disclosure of Potential Conflicts of InterestNo potential
conflicts of interest were disclosed.
Authors' ContributionsConception and design: J. Liu, W.L.
McKeehan, C. Wang, W. Zhong,F. WangDevelopment of methodology: J.
Liu, G. Chen, Z. Liu, S. Liu, Z. Cai, P. You,Y. Ke, Y. Huang, H.
Pelicano, C.-L. Wu, C. Wang, W. Zhong, F. WangAcquisition of data
(provided animals, acquired and managed patients,provided
facilities, etc.): J. Liu, G. Chen, Z. Liu, S. Liu, Z. Cai, P. You,
Y. Ke,L. Zhao, H. Pelicano, C.-L. WuAnalysis and interpretation of
data (e.g., statistical analysis, biostatistics,computational
analysis): J. Liu, G. Chen, Z. Liu, S. Liu, Z. Cai, P. You, Y.
Ke,L. Lai, Y. Huang, H. Gao, L. Zhao, H. Pelicano, P. Huang, W.L.
McKeehan,C.-L. Wu, C. Wang, W. Zhong, F. WangWriting, review,
and/or revision of the manuscript: J. Liu, G. Chen, Z. Liu,S. Liu,
Z. Cai, P. You, Y. Ke, Y. Huang, H. Gao, P. Huang, C.-L. Wu, C.
Wang,W. Zhong, F. WangAdministrative, technical, or material
support (i.e., reporting or organizingdata, constructing
databases): J. Liu, C. Wang, W. Zhong, F. WangStudy supervision:
W.L. McKeehan, C.-L. Wu, C. Wang, W. Zhong, F. Wang
AcknowledgmentsWe thank Drs. Juha Patanen and David Ornitz for
sharing the Fgfr1floxed and
Fgfr2floxed mice, respectively. This work was supported by the
NIH CA96824,TAMU1400302, CPRIT 110555 grants, and a gift from
Agilent Technology, Inc.(to F. Wang), the National Natural Science
Foundation of China 81101712,31371470, and 81270761 grants (to
C.Wang); andNational Key Basic ResearchProgram of China
(2015CB553706), National Natural Science Foundation ofChina
(81571427) and the Fundamental Research Funds for the
CentralUniversities 2017PY023 (to W.D. Zhong).
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 October 20, 2017; revised March 29, 2018; accepted June
4, 2018;published first June 11, 2018.
Liu et al.
Cancer Res; 78(16) August 15, 2018 Cancer Research4468
on June 22, 2021. © 2018 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
Published OnlineFirst June 11, 2018; DOI:
10.1158/0008-5472.CAN-17-3226
http://cancerres.aacrjournals.org/
-
References1. Fischer K,HoffmannP, Voelkl S,MeidenbauerN, Ammer
J, EdingerM, et al.
Inhibitory effect of tumor cell-derived lactic acid on human T
cells. Blood2007;109:3812–9.
2. Brand A, Singer K, Koehl GE, Kolitzus M, Schoenhammer G,
Thiel A, et al.LDHA-associated lactic acid production blunts tumor
immunosurveillanceby T and NK cells. Cell Metab 2016;24:657–71.
3. Ciccarese C, Santoni M, Massari F, Modena A, Piva F, Conti A,
et al.Metabolic alterations in renal and prostate cancer. Curr Drug
Metab2015;17:150–5.
4. Xian ZY, Liu JM, ChenQK, ChenHZ, Ye CJ, Xue J, et al.
Inhibition of LDHAsuppresses tumor progression in prostate cancer.
Tumour Biol 2015;36:8093–100.
5. Pertega-Gomes N, Felisbino S, Massie CE, Vizcaino JR, Coelho
R, Sandi C,et al. A glycolytic phenotype is associated with
prostate cancer progressionand aggressiveness: a role for
monocarboxylate transporters as metabolictargets for therapy. J
Pathol 2015;236:517–30.
6. Mraz J, Vrubel F,HanselovaM. Carcinoma of the prostate. II.
Serum activityof acid phosphatase, prostatic acid phosphatase, LDH
and its isoenzymes.Int Urol Nephrol 1979;11:301–9.
7. Vrubel F, Mraz J, Nemecek R, Papousek F, Hanselova M.
Carcinoma of theprostate. I. Histochemical examination as an aid in
evaluating prostatecarcinoma. Int Urol Nephrol 1979;11:295–9.
8. Oliver JA, el-Hilali MM, Belitsky P, MacKinnon KJ. LDH
isoenzymes inbenign and malignant prostate tissue. The LDH V-I
ratio as an index ofmalignancy. Cancer 1970;25:863–6.
9. Naruse K, Yamada Y, Aoki S, Taki T, Nakamura K, TobiumeM, et
al. Lactatedehydrogenase is a prognostic indicator for prostate
cancer patients withbone metastasis. Hinyokika Kiyo
2007;53:287–92.
10. Keshari KR, SriramR, VanCriekingeM,WilsonDM,Wang ZJ,
VigneronDB,et al. Metabolic reprogramming and validation of
hyperpolarized 13Clactate as a prostate cancer biomarker using a
human prostate tissue sliceculture bioreactor. Prostate
2013;73:1171–81.
11. Wang F, Luo Y, McKeehan W. The FGF signaling axis in
prostate tumor-igenesis. In: E. G, C. S, F. R, editors. Molecular
oncology: causes of cancerand targets for treatment.
London,UnitedKingdom:CambridgeUniversityPress; 2013. p. 186–9.
12. Zhang Y, Zhang J, Lin Y, Lan Y, Lin C, Xuan JW, et al. Role
of epithelialcell fibroblast growth factor receptor substrate
2{alpha} in prostatedevelopment, regeneration and tumorigenesis.
Development 2008;135:775–84.
13. Yang F, ZhangY, Ressler SJ, IttmannMM,AyalaGE,Dang TD, et
al. FGFR1 isessential for prostate cancer progression and
metastasis. Cancer Res2013;73:3716–24.
14. Yu P, Wilhelm K, Dubrac A, Tung JK, Alves TC, Fang JS, et
al. FGF-dependent metabolic control of vascular development. Nature
2017;545:224–8.
15. Fritz PJ. Rabbit muscle lactate dehydrogenase 5; a
regulatory enzyme.Science 1965;150:364–6.
16. Semenza GL, Jiang BH, Leung SW, Passantino R, Concordet
JP,Maire P, et al. Hypoxia response elements in the aldolase A,
enolase1, and lactate dehydrogenase A gene promoters contain
essentialbinding sites for hypoxia-inducible factor 1. J Biol Chem
1996;271:32529–37.
17. Liang X, Liu L, Fu T, ZhouQ, ZhouD, Xiao L, et al. Exercise
inducible lactatedehydrogenase B regulates mitochondrial function
in skeletal muscle.J Biol Chem 2016;291:25306–18.
18. Dennison JB, Molina JR, Mitra S, Gonzalez-Angulo AM, Balko
JM, KubaMG, et al. Lactate dehydrogenase B: a metabolic marker of
response toneoadjuvant chemotherapy in breast cancer. Clin Cancer
Res 2013;19:3703–13.
19. McCleland ML, Adler AS, Deming L, Cosino E, Lee L,
BlackwoodEM, et al. Lactate dehydrogenase B is required for the
growth ofKRAS-dependent lung adenocarcinomas. Clin Cancer Res
2013;19:773–84.
20. McClelandML, Adler AS, Shang Y, Hunsaker T, Truong T,
Peterson D, et al.An integrated genomic screen identifies LDHB as
an essential gene fortriple-negative breast cancer. Cancer Res
2012;72:5812–23.
21. Zha X,Wang F,Wang Y,He S, Jing Y,Wu X, et al. Lactate
dehydrogenase B iscritical for hyperactive mTOR-mediated
tumorigenesis. Cancer Res2011;71:13–8.
22. Maekawa M, Taniguchi T, Ishikawa J, Sugimura H, Sugano K,
Kanno T.Promoter hypermethylation in cancer silences LDHB,
eliminating lactatedehydrogenase isoenzymes 1–4. Clin Chem
2003;49:1518–20.
23. Leiblich A, Cross SS, Catto JW, Phillips JT, Leung HY, Hamdy
FC, et al.Lactate dehydrogenase-B is silenced by promoter
hypermethylation inhuman prostate cancer. Oncogene
2006;25:2953–60.
24. Cui J, Quan M, Jiang W, Hu H, Jiao F, Li N, et al.
Suppressed expression ofLDHB promotes pancreatic cancer progression
via inducing glycolyticphenotype. Med Oncol 2015;32:143.
25. Kim JH, Kim EL, Lee YK, Park CB, Kim BW, Wang HJ, et al.
Decreasedlactate dehydrogenase B expression enhances claudin
1-mediatedhepatoma cell invasiveness via mitochondrial defects. Exp
Cell Res2011;317:1108–18.
26. Chen R, Zhou X, Yu Z, Liu J, Huang G. Low expression of LDHB
correlateswith unfavorable survival in hepatocellular carcinoma:
strobe-compliantarticle. Medicine 2015;94:e1583.
27. Brisson L, Banski P, Sboarina M, Dethier C, Danhier P,
Fontenille MJ, et al.Lactate dehydrogenase B controls lysosome
activity and autophagy incancer. Cancer Cell 2016;30:418–31.
28. Li SS, Pan YE, Sharief FS, Evans MJ, Lin MF, Clinton GM, et
al. Cancer-associated lactate dehydrogenase is a
tyrosylphosphorylated formof human LDH-A, skeletal muscle
isoenzyme. Cancer Invest 1988;6:93–101.
29. Fan J, Hitosugi T, Chung TW, Xie J, Ge Q, Gu TL, et al.
Tyrosine phos-phorylation of lactate dehydrogenase A is important
for NADH/NAD(þ)redox homeostasis in cancer cells. Mol Cell Biol
2011;31:4938–50.
30. Lin Y, Liu G, Zhang Y, Hu YP, Yu K, Lin C, et al. Fibroblast
growth factorreceptor 2 tyrosine kinase is required for prostatic
morphogenesis and theacquisition of strict androgen dependency for
adult tissue homeostasis.Development 2007;134:723–34.
31. Wang C, Chang JY, Yang C, Huang Y, Liu J, You P, et al. Type
1 fibroblastgrowth factor receptor in cranial neural crest
cells-derived mesenchyme isrequired for palatogenesis. J Biol Chem
2013;288:22174–83.
32. Huang Y, Jin C, Hamana T, Liu J, Wang C, An L, et al.
Overexpressionof FGF9 in prostate epithelial cells augments
reactive stroma forma-tion and promotes prostate cancer
progression. Int J Biol Sci 2015;11:948–60.
33. Zhong WD, Liang YX, Lin SX, Li L, He HC, Bi XC, et al.
Expression ofCD147 is associated with prostate cancer progression.
Int J Cancer2012;130:300–8.
34. Nishitani S, Takehana K, Fujitani S, Sonaka I.
Branched-chain amino acidsimprove glucose metabolism in rats with
liver cirrhosis. Am J PhysiolGastrointest Liver Physiol
2005;288:G1292–300.
35. Acevedo VD, Gangula RD, Freeman KW, Li R, Zhang Y, Wang F,
et al.Inducible FGFR-1 activation leads to irreversible prostate
adenocarci-noma and an epithelial-to-mesenchymal transition. Cancer
Cell 2007;12:559–71.
36. Ozen M, Giri D, Ropiquet F, Mansukhani A, Ittmann M. Role of
fibroblastgrowth factor receptor signaling in prostate cancer cell
survival. J NatlCancer Inst 2001;93:1783–90.
37. Abate-Shen C, Shen MM. FGF signaling in prostate
tumorigenesis–newinsights into epithelial–stromal interactions.
Cancer Cell 2007;12:495–7.
38. Taylor BS, Schultz N, Hieronymus H, Gopalan A, Xiao Y,
Carver BS, et al.Integrative genomic profiling of human prostate
cancer. Cancer Cell2010;18:11–22.
39. Giri D, Ropiquet F, IttmannM. Alterations in expression of
basic fibroblastgrowth factor (FGF) 2 and its receptor FGFR-1 in
human prostate cancer.Clin Cancer Res 1999;5:1063–71.
40. Devilard E, Bladou F, RamuzO, KarsentyG,Dales JP, Gravis G,
et al. FGFR1and WT1 are markers of human prostate cancer
progression. BMC Cancer2006;6:272.
41. Wang J, Stockton DW, Ittmann M. The fibroblast growth factor
receptor-4Arg388 allele is associated with prostate cancer
initiation and progression.Clin Cancer Res 2004;10:6169–78.
42. Sugiura K, Su YQ, Diaz FJ, Pangas SA, Sharma S, Wigglesworth
K, et al.Oocyte-derived BMP15 and FGFs cooperate to promote
glycolysis incumulus cells. Development 2007;134:2593–603.
43. Jin L, Chun J, Pan C, Alesi GN, Li D, Magliocca KR, et al.
Phosphorylation-mediated activation of LDHA promotes cancer cell
invasion and tumourmetastasis. Oncogene 2017;36:3797–806.
FGF Signaling in Cell Energy Metabolism
www.aacrjournals.org Cancer Res; 78(16) August 15, 2018 4469
on June 22, 2021. © 2018 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
Published OnlineFirst June 11, 2018; DOI:
10.1158/0008-5472.CAN-17-3226
http://cancerres.aacrjournals.org/
-
44. Zhao D, Zou SW, Liu Y, Zhou X, Mo Y, Wang P, et al. Lysine-5
acetylationnegatively regulates lactate dehydrogenase A and is
decreased in pancreaticcancer. Cancer Cell 2013;23:464–76.
45. Koh YW, Lee SJ, Park SY. Prognostic significance of lactate
dehydroge-nase B according to histologic type of non-small-cell
lung cancer and its
association with serum lactate dehydrogenase. Pathol Res Pract
2017;213:1134–8.
46. Li C, Chen Y, Bai P, Wang J, Liu Z, Wang T, et al. LDHBmay
be a significantpredictor of poor prognosis in osteosarcoma. Am J
Transl Res 2016;8:4831–43.
Cancer Res; 78(16) August 15, 2018 Cancer Research4470
Liu et al.
on June 22, 2021. © 2018 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
Published OnlineFirst June 11, 2018; DOI:
10.1158/0008-5472.CAN-17-3226
http://cancerres.aacrjournals.org/
-
2018;78:4459-4470. Published OnlineFirst June 11, 2018.Cancer
Res Junchen Liu, Guo Chen, Zezhen Liu, et al. Prostate CancerEffect
by Reprogramming LDH Isoform Expression and Activity in Aberrant
FGFR Tyrosine Kinase Signaling Enhances the Warburg
Updated version
10.1158/0008-5472.CAN-17-3226doi:
Access the most recent version of this article at:
Material
Supplementary
http://cancerres.aacrjournals.org/content/suppl/2018/10/18/0008-5472.CAN-17-3226.DC1
Access the most recent supplemental material at:
Overview
Visual
http://cancerres.aacrjournals.org/content/78/16/4459/F1.large.jpgA
diagrammatic summary of the major findings and biological
implications:
Cited articles
http://cancerres.aacrjournals.org/content/78/16/4459.full#ref-list-1
This article cites 45 articles, 17 of which you can access for
free at:
E-mail alerts related to this article or journal.Sign up to
receive free email-alerts
Subscriptions
Reprints and
[email protected]
To order reprints of this article or to subscribe to the
journal, contact the AACR Publications Department at
Permissions
Rightslink site. Click on "Request Permissions" which will take
you to the Copyright Clearance Center's (CCC)
.http://cancerres.aacrjournals.org/content/78/16/4459To request
permission to re-use all or part of this article, use this link
on June 22, 2021. © 2018 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
Published OnlineFirst June 11, 2018; DOI:
10.1158/0008-5472.CAN-17-3226
http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-17-3226http://cancerres.aacrjournals.org/content/suppl/2018/10/18/0008-5472.CAN-17-3226.DC1http://cancerres.aacrjournals.org/content/78/16/4459/F1.large.jpghttp://cancerres.aacrjournals.org/content/78/16/4459.full#ref-list-1http://cancerres.aacrjournals.org/cgi/alertsmailto:[email protected]://cancerres.aacrjournals.org/content/78/16/4459http://cancerres.aacrjournals.org/
/ColorImageDict > /JPEG2000ColorACSImageDict >
/JPEG2000ColorImageDict > /AntiAliasGrayImages false
/CropGrayImages false /GrayImageMinResolution 200
/GrayImageMinResolutionPolicy /Warning /DownsampleGrayImages true
/GrayImageDownsampleType /Bicubic /GrayImageResolution 300
/GrayImageDepth -1 /GrayImageMinDownsampleDepth 2
/GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true
/GrayImageFilter /DCTEncode /AutoFilterGrayImages true
/GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict >
/GrayImageDict > /JPEG2000GrayACSImageDict >
/JPEG2000GrayImageDict > /AntiAliasMonoImages false
/CropMonoImages false /MonoImageMinResolution 600
/MonoImageMinResolutionPolicy /Warning /DownsampleMonoImages true
/MonoImageDownsampleType /Bicubic /MonoImageResolution 900
/MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000
/EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode
/MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None
] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false
/PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000
0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true
/PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ]
/PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier ()
/PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped
/False
/CreateJDFFile false /Description > /Namespace [ (Adobe)
(Common) (1.0) ] /OtherNamespaces [ > /FormElements false
/GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks
false /IncludeInteractive false /IncludeLayers false
/IncludeProfiles false /MarksOffset 18 /MarksWeight 0.250000
/MultimediaHandling /UseObjectSettings /Namespace [ (Adobe)
(CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /NA
/PageMarksFile /RomanDefault /PreserveEditing true
/UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling
/LeaveUntagged /UseDocumentBleed false >> > ]>>
setdistillerparams> setpagedevice