[5] Zhang et all Cell

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Glycine Decarboxylase Activity Drives

Non-Small Cell Lung CancerTumor-Initiating Cells and TumorigenesisWen Cai Zhang,1,3 Ng Shyh-Chang,1 He Yang,5 Amit Rai,6 Shivshankar Umashankar,6,7 Siming Ma,1 Boon Seng Soh,1

Li Li Sun,1 Bee Choo Tai,11 Min En Nga,9 Kishore Kumar Bhakoo,12 Senthil Raja Jayapal,13 Massimo Nichane,1 Qiang Yu,2

Dokeu A. Ahmed,4 Christie Tan,4 Wong Poo Sing,10 John Tam,10Agasthian Thirugananam,14 Monireh Soroush Noghabi,1

Yin Huei Pang,9 Haw Siang Ang,5 Wayne Mitchell,16,17 Paul Robson,1 Philipp Kaldis,13 Ross Andrew Soo,5,8

Sanjay Swarup,6,7 Elaine Hsuen Lim,3,8,15,*and Bing Lim1,18,*1Stem Cell and Developmental Biology2Cancer Biology and Pharmacology

Genome Institute of Singapore, 60 Biopolis Street, Singapore 1386723Department of Respiratory Medicine4Department of Thoracic Surgery

Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, Singapore 3084335Cancer Science Institute of Singapore, National University of Singapore, 28 Medical Drive, Singapore 1174566Metabolites Biology Lab, Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 1175437NUS Environmental Research Institute (NERI), T-Lab Building (TL), National University of Singapore, 5A Engineering Drive 1, Singapore 1174118Department of Haematology-Oncology9Department of Pathology10Department of Thoracic Surgery

National University Hospital, 5 Lower Kent Ridge Road, Singapore 11907411Department of Community, Occupational and Family Medicine, Yong Loo Lin School of Medicine, National University of Singapore,

Singapore 11759712Translational Molecular Imaging Group (TMIG), Laboratory of Molecular Imaging (LMI), Singapore BioImaging Consortium (SBIC),

11 Biopolis Way, Singapore 13866713Cell Division and Cancer Laboratory (PRK), Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 13867314Department of Thoracic Surgery15Department of Medical Oncology

National Cancer Centre, 11 Hospital Drive, Singapore 169610

16Bioinformatics, Experimental Therapeutics Centre, 31 Biopolis Way, Singapore 13866917Divisionof InformationSciences, Schoolof Computer Engineering, NanyangTechnological University, 50 NanyangAvenue, Singapore 63979818Beth-Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA

*Correspondence: [email protected](E.H.L.),[email protected](B.L.)

DOI10.1016/j.cell.2011.11.050

SUMMARY

Identification of the factors critical to the tumor-initi-

ating cell (TIC) state mayopen new avenues in cancer

therapy. Here we show that the metabolic enzyme

glycine decarboxylase (GLDC) is critical for TICs in

non-small cell lung cancer (NSCLC). TICs from pri-mary NSCLC tumors express high levels of the onco-

genic stem cell factor LIN28B and GLDC, which are

both required for TIC growth and tumorigenesis.

Overexpression of GLDC and other glycine/serine

enzymes, but not catalytically inactive GLDC, pro-

motes cellular transformation and tumorigenesis.

We found that GLDC induces dramatic changes in

glycolysis and glycine/serine metabolism, leading

to changes in pyrimidine metabolism to regulate

cancer cell proliferation. In the clinic, aberrant activa-

tion of GLDC correlates with poorer survival in lung

cancer patients, and aberrant GLDC expression is

observed in multiple cancer types. This link between

glycine metabolism and tumorigenesis may provide

novel targets for advancing anticancer therapy.

INTRODUCTION

Despite numerous advances in our knowledge of cancer (Vogel-

stein and Kinzler, 2004; Hanahan and Weinberg, 2011), our

ability to develop clinically effective therapies based on this

understanding has met with limited success. Current therapies

can control tumor growth initially, but most patients ultimately

relapse. One prominent example is lung cancer, the leading

cause of cancer-related mortality with over 1 million deaths

each year (Jemal et al., 2011). Non-small cell lung cancer

(NSCLC) accounts for approximately 85% of all lung cancers.

Although NSCLC patients with epidermal growth factor receptor

(EGFR) mutations respond to EGFR inhibitors initially, most

patients experience a relapse within 1 year (Sequist et al.,

Cell148, 259272, January 20, 2012 2012 Elsevier Inc. 259
mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.cell.2011.11.050http://dx.doi.org/10.1016/j.cell.2011.11.050mailto:[email protected]:[email protected]


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2007). These findings underscore the urgent need for both com-

bination therapies and also new approaches to treat cancerous

tumors. One such approach may be to target tumor-initiating

cells (TICs).

Data from leukemias, germ cell tumors, and a number of solidtumors support the notion that cancers are maintained by

a subpopulation of self-renewing and evolving TICs. This is

also popularly known as the cancer stem cell (CSC) model

(Reya et al., 2001; Rosenand Jordan, 2009). Although the validity

of the CSC model is an issue of controversy in melanoma (Quin-

tana et al., 2008, 2010; Boiko et al., 2010; Civenni et al., 2011),

many other solid tumors appear to follow the CSC model (Ishi-

zawa et al., 2010). Recently it was proposed that at earlier stages

of tumorigenesis, rare TIC clones differentiate into nonmalignant

progeny to form the bulk of the tumor, whereas at advanced

stages, TIC clones constitute the bulk of the tumor (Boiko

et al., 2010). Studies with mouse models of lung cancer have

also begun to reconcile the connection between the evolving

genotype of TIC clones and the surface phenotype of TICs (Cur-tis et al., 2010). Thus accumulated findings suggest that target-

ing TICs may be a promising approach for eradicating tumors

early. However, progress in the targeting of TICs to improve

cancer therapy has been hindered by a lack of understanding

of the molecular pathways that are critical to TICs.

Recent studies have led to an emerging appreciation of the

importance of metabolic reprogramming in cancer (Hsu and Sa-

batini, 2008; Vander Heiden et al., 2009; Hanahan andWeinberg,

2011). Most recently, the embryonic isoform of pyruvate kinase

PKM2, in collaboration with phosphoglycerate mutase, was

found to regulate the shift from oxidative phosphoryla-

tion to glycolysis in cancer cells (Christofk et al., 2008; Vander

Heiden et al., 2010). These findings have led to a resurgence of

interest in the Warburg effectthe phenomenon whereby cancer

cells, like embryonic cells, preferentially use glycolysis even

under aerobic conditions (Warburg, 1956). Besides glycolysis,

an arm of metabolism that results in sarcosine production has

also been implicated in prostate cancer (Sreekumar et al.,

2009). These data suggest that metabolic reprogramming is

crucial for tumorigenesis, and much remains to be uncovered.

Here we show that glycine metabolism and the metabolic

enzyme glycine decarboxylase (GLDC) drive TICs and tumori-

genesis in NSCLC. Using CD166 as a surface marker and

NOD/SCID Il2rg/ mice as xenotransplantation recipients, we

isolated lung TICs from a broad range of primary NSCLC tumors

(stages IIII). Primary lung TICs express high levels of LIN28B,

GLDC, and many other glycine/serine metabolism enzymes.Both LIN28B and GLDC were required for lung TIC proliferation

and tumor growth. Overexpression of GLDC alone, and other

glycine/serine enzymes, promotes cellular transformation both

in vitro andin vivo. Metabolomic analysis shows that GLDC over-

expression induces dramatic changes in glycolysis and glycine

metabolism, leading to changes in pyrimidine metabolism for

cancer cell proliferation. In human patients, aberrant upregula-

tion of GLDC is significantly associated with higher mortality

from lung cancer, and aberrant GLDC expression is observed

in multiple cancer types. Our findings establish a link between

glycine metabolism and tumorigenesis and may provide novel

targets for advancing anticancer therapy.

RESULTS

TICs in Lung Cancer

To assess the cellular heterogeneity within NSCLC, we obtained

freshly resected lung tumors from 36 human patients with abroad range of stage IIII primary NSCLC (Table S1 available

online). Patient lung cancer cells were directly transplanted

subcutaneously into NOD/SCID Il2rg/ mice with Matrigel

(Quintana et al., 2008). Using this maximally sensitive assay,

we estimated by limiting dilution analysis that lung TICs exist

with a low frequency of 1 in 4 3 105 cells in unsorted NSCLC

tumor cells(n = 36 patients; Figure1A), consistent with published

findings (Ishizawa et al., 2010).

To profile the surface phenotype of this subpopulation of lung

TICs, we fractionated the NSCLC tumors by fluorescence-

activated cell sorting (FACS; Figure S1A). After excluding

hematopoietic and endothelial cells (Lin), we tested a panel of

cell-surface markers, including CD166, CD44, CD133, and

EpCAM (Figure 1B). We found that CD166 was the most robustmarker for enriching the lung TIC subpopulation, compared to

CD133, CD44, or EpCAM, allowing us to reliably enrich lung

TICs by nearly 100-fold (Figures 1A and 1B). In 12 out of 12

NSCLC patient tumors (lung adenocarcinoma), the CD166+

Lin fraction contained cells that consistently initiated lung tumor

formation in vivo. In contrast, CD166Lin tumor cells generally

failed to initiate lung tumor formation even after 8 months of

observation, although they also expressed carcinoembryonic

antigen (CEA), a tumor-specific marker not expressed in normal

adult lung cells (Figures 1A, 1B, andS1B). Similar results were

observed in lung squamous cell carcinoma and large cell carci-

noma (Figure S1C). Although CD166 expression varied across

the NSCLC tumors we examined, CD166 was consistently

higher in lung tumors than in normal adjacent lung tissues

(n = 25 patients;Figures S1D and S1E).

CD166+ lung TICs demonstrate a capacity for self-renewal

and differentiation in vivo. Serial transplantations showed that

only the CD166+ fraction was able to self-renew and initiate pri-

mary and secondary xenograft tumors (Figures 1A and S1F).

Upon transplantation, CD166+ lung TICs differentiated to form

xenograft tumors that phenocopy the complex cytoarchitec-

ture of their parental patient tumors, sharing similar histolog-

ical morphology by hematoxylin-eosin (H&E) staining and

similar tissue distributions of CD166, cytokeratin, E-cadherin,

vimentin, smooth muscle actin, and synaptophysin (Figures

1C, S1G, and S1H). Furthermore, we found that transplants

with more TICs grow more rapidly, suggesting that lung TICfrequency is correlated with tumor growth rate (Figures 1D

andS1I).

The self-renewal capacity of CD166+ lung TICs is further

corroborated by in vitro assays. We tested the CD166+ fraction

for the ability to form tumor spheres, a widely used in vitro tech-

nique for assessing self-renewal capacity (Dontu et al., 2003).

Although both primary CD166+ and CD166 cells remained

viable in vitro, only primary CD166+ but not CD166 cells were

able to form compact self-renewing spheres (n = 9 patients;

Figures 1E, 1F, andS1J). Using immunofluorescence and flow

cytometry, we found that the lung tumor spheres retained high

levels of CD166 expression but undetectable CD133 expression

260 Cell148, 259272, January 20, 2012 2012 Elsevier Inc.


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in contrast (Figures S1K and S1L). When primary lung tumor

spheres were dissociated into single cells and transplanted

into NOD/SCID Il2rg/ mice in vivo, we found that as few as

15 single cells consistently initiated tumorigenesis (Figures 1G

andS1M).

The increased tumor-initiating frequency of lung tumor sphere

cells suggests that they are even more highly enriched for lung

TICs than the patient tumor CD166+ fraction, and that lung

TICs expanded during in vitro culture to form tumor spheres.

To test whether CD166 drives tumorigenicity in lung TICs, we

A

CellsNo. cells per

injection

No. tumors

/ No.

injections

LTICs

frequency

(95% CI)

Enrichment

factor (x)Pvalue

150,000 2/4

C

H&E

Patient tumor Xenograft tumor

Unsorted

1/403,971

(1/255,949 -

1/637,598)

1

200,000 1/10

300,000 4/5

500,000 3/5

1,000,000 9/9

2,000,000 2/2

CD166+Lin-1/5,175

(1/2,837 -

1/9,441)

78 1.37E-23

1,000 2/8

5,000 2/3

10,000 7/9

23,000 2/2

25 000 4/4

CD166

PopulationNo. tumors/

No. injections

CD166+ 12/12

B

,

50,000 1/1

CD166-Lin-1/3,297,091

(1/460,464 -

1/23,608,384)

0.1 4.82E-03210,000 0/1

260,000 1/3

500,000 0/3

400

mm3)

CD166+Lin- (1st xenograft; 5,000)

CD166+Lin- (patient; 5,000)

Lin- (patient; 150,000)

D

pan-CK

CD166- 0/10

CD44+ 6/8

CD44- 6/8

CD133+ 6/11

CD133- 7/13

EpCAM+ 10/13

EpCAM- 8/14 0

100

200

300

14 15 16 17 18 19 20 21

Tumorvolume

Weeks

CD166-L n- (pat ent; 100,000)

AdC

F G

0

30

60

90

120

No.ofspheres/10,0

00cells

AdC SCC LCC

E

D166-Lin-

SCCAdC LCC

CD166+Lin-

No.

Cells

No. tumors/ No. injections

1st passage 10th passage

2500 12/12 N.D.

500 18/18 18/18

50 24/24 24/24

10 12/12 12/12

5 16/18 17/18

1 5/18 5/18

GTumor sphere assay

CD

Figure 1. CD166+ Fraction Contains TICs from NSCLC Patients

(A)Frequency of TICs in unsorted,CD166+, andCD166 subpopulationsof cells from36 NSCLCpatients. CI, confidence interval. Lin cells, CD45 CD31 cells.

(B) Tumor initiation frequency by various FACS-purified fractions of Lin lung cancer cells isolated from primary xenograft tumors shown in (A). 5 3 104

CD166,

CD44, CD133, and EpCAM cells were tested for tumor initiation in NOD/SCID Il2rg/ mice (n = 12).

(C) Histological analysis of patient tumors and primary xenografts derived from patient tumor CD166+

Lin lung cancer cells. Tumors were stained for H&E,

CD166, and pan-CK (cytokeratin). Scale bar, 50 mm.

(D) Representative tumor-growth curves of xenografts derived from different cell fractions in a lung adenocarcinoma (AdC) patient tumor and the primaryxenograft.

(E) Phase-contrast images of tumor spheres seeded with CD166+ Lin (top) and CD166 Lin (bottom) cells in lung adenocarcinoma (AdC), squamous cell

carcinoma (SCC), and large cell carcinoma (LCC). Scale bar, 50 mm.

(F) Quantification of tumor spheres formed by cells from NSCLC patient (AdC, SCC, or LCC) CD166+Lin, CD166Lin, and Lin populations.

(G) Frequency of tumorigenesis by single patient-derived tumor sphere cells (n = 3). N.D., not determined.

In all panels, error bars represent the standard error of the mean (SEM). See alsoFigure S1andTable S1.



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knocked down CD166 in two lines of NSCLC patient-derived

tumor spheres by retroviral shRNA (Figure S1N). We found that

the tumorigenicity of lung TICs in the tumor spheres was not

significantly affected by CD166 shRNA, demonstrating that

CD166 is an inert cell-surface marker that enriches for lung

TICs (Figures S1OS1Q).

Lung TICs Express High levels of Glycine/Serine

Metabolism Enzymes

To gain a deeper understanding of the molecular basis for the

TIC state and its tumorigenic capacity, we sought to obtain

a molecular signature for lung TICs. To do this, we performed

genome-wide transcriptome analysis on CD166 Lin tumor

cells, CD166+ Lin tumor cells, and lung tumor spheres, in

increasing order of lung TIC frequency (Figure 2A). As a negative

control, we also profiled CD166+ versus CD166 cells from

normal adjacent lung tissues (n = 3 patients; Table S1). This

led us to a profile of genes that are upregulated and down-

regulated in lung TICs, compared to non-TICs (Figure 2B).

Lung TIC-associated genes include the oncogenic stem cell

factor LIN28B, embryonic lung transcription factors like PEA3

and thetrachealesshomologNPAS1 (Viswanathan et al., 2009;

4

o

4

BA

Enrichment

S/X-

X+/X-

P+/P-

N+/N-

Log2ratio

0

-4N+/X- P+/X- S/X-X+/X-

Log2rati

0

-4N+/X- P+/X- S/X-X+/X_

X+ S

Candidate

genes

S: sphere; X, xenograft tumor; P, primary tumor;

N, normal tissue; +, CD166+; -, CD166-.

1

2

3

4

onlevel(log2ratio)

S/X+ X+/X-

P+/P- N+/N-

HSPA1A

IGLL1

RENB

P

PPP1R

15A

RASD

1

MMP9

RRAD

CXCL

2

GADD

45G

TNFAIP3

C D

0% 2% 4% 6% 8% 10%

Cell cycle

DNA replication

Percentage of genes in pathway

-5

-4

-3

-2

-1

RelativemRNAexpressio

GLDC

NPAS1

LIN28B

RG9MTD2

CCNB1

TMEM118

SMC6

PEA3

REEP1

FOXK2

Glycine, serine and threonine

metabolism

Pyrimidine metabolism

MAPK signaling pathway

p53 signaling pathwayExperimental

Predicted

E

PSPHGlycine CO2 + NH3 + CH2Phosphoserine Serine

GLDC

L-2-Amino-acetoacetate

GCAT

SHMT13P-Hydroxy pyruvate

PSAT1

Figure 2. Lung TICs Express High Levels of GLDC and LIN28B

(A) Venndiagramshowingstrategy for enriching tumorigenic geneexpressionprofile by genome-wide transcriptomeanalysis.A listof thedifferentially expressed

genes(cutoff thresholdof 1.5-fold,p < 0.05)common between P+ versusP, X+ versus X, and S versusXwasderived, withthe differentially expressed genesof

N+

versus N excluded. Thegenelistwas further filtered to select only genesfurtherupregulatedor downregulated in S versus X+

. N,normal lungtissue (n= 3); P,

patient tumor (n = 1); S, tumor sphere (n = 4); X, xenograft tumor (n = 3); +, CD166+

; , CD166. Total, n = 11.

(B) Graphs of relativeexpression of candidatelung TIC-associated genes in increasing (left, n = 194) or decreasing (right, n = 295) trends across differentCD166+

fraction cells from normal lung tissue (N), primary tumor (P), xenograft tumor (X), and tumor spheres (S) versus nontumorigenic CD166 (X) cells.

(C) Top-ranked genes differentially expressed in lung TICs.

(D) Enrichment of KEGG pathways by genes differentially expressed in lung TICs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

(E) Schematic diagram of glycine, serine, and threonine metabolism genes significantly (p < 0.05) upregulated in lung TICs. [, upregulation.

See alsoFigure S2.



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Liu et al., 2003; Levesque et al., 2007), as well as cell-cycle regu-

lators like CCNB1 and GADD45G (Figure 2C). The highest-

ranking genes were validated by qRT-PCR (Figure S2A). KEGG

pathway analysis of the lung TIC-gene profile showed that the

top enriched pathways were cell cycle, DNA replication,glycine, serine, and threonine metabolism, pyrimidine metab-

olism, MAPK signaling pathway, and p53 signaling path-

way (Figure 2D). Within the glycine, serine, and threonine

metabolism pathway, we found that glycine/serine metabolism

enzymes likeGLDC, glycine C-acetyltransferase (GCAT), serine

hydroxymethyltransferase (SHMT1), phosphoserine phospha-

tase (PSPH), and phosphoserine aminotransferase (PSAT1)

were all upregulated in lung TICs (Figures 2E andS2BS2D). In

particular,GLDCwas one of the most highly upregulated genes

in multiple analyses of lung TIC-enriched populations, at both the

mRNA and protein levels (Figures 2C andS2C). GLDC is a key

component of the highly conserved glycine cleavage system

in amino acid metabolism that catalyzes the breakdown of

glycine to form CO2, NH3, and 5,10-methylene-tetrahydrofolate(CH2-THF) to fuel one-carbon metabolism (Kume et al., 1991).

GLDC Is an Oncogene that Promotes Tumorigenesis

and Cellular Transformation

High expression ofGLDCandLIN28Bin lung TIC-enriched pop-

ulations,but not in CD166 lung cancer cells and CD166+ normal

lung cells, suggests that these two genes drive tumorigenicity in

lung TICs. To test this hypothesis, we knocked down GLDCand

LIN28B in lung tumor spheres with shRNAs (Figure S3A) and

compared their growth both in vitro and in vivo. We found that

both GLDC and LIN28B were necessary for cell proliferation in

sphere culture, as well as anchorage-independent colony forma-

tionin softagar (Figures 3A and S3B). Importantly, tumorigenicity

was also significantly reduced upon knockdown of either GLDC

or LIN28B (Figures 3B and S3C). A549 lung adenocarcinoma

cells showed similar results (Figures S3DS3G). Our results

suggest that lung TICs and lung tumorigenesis are dependent

on GLDC. This led us to ask what oncogenes upregulate

GLDC. Because the E2F pathway upregulates many metabolic

genes during cell proliferation, we examined the expression of

GLDC over the course of the cell cycle in both normal human

lung fibroblasts (HLFs) and the transformed A549 cells after

synchronization by serum starvation. Our results showed that

GLDC is insensitive to cell-cycle progression in both normal

HLFs and transformed A549 cells, suggesting that GLDC is not

regulated by cell-cycle or E2F signals (Figure 3C). We then

examinedGLDClevels in MCF10A cells after transformation byoncogenic KRASG12D, PIK3CAE545K, and MYCT58A. Our results

show that all three oncogenes induceGLDC by 20-fold, sug-

gesting that oncogene-induced GLDC transcription is common

to the cellular transformation process mediated by oncogenic

Ras, PI3K, and Myc (Figure 3D).

To test whether aberrant GLDC upregulation is sufficient

to drive cellular transformation, as has been shown for LIN28B

(Viswanathan et al., 2009), we overexpressed GLDC in NIH/

3T3 cells (Figure S3H). We found that GLDC overexpression

significantly increased colony formation by 3T3 cells under nor-

mal culture conditions (Figures 3E and 3F). To test for cellular

transformation in vitro, we cultured the 3T3 cells overexpressing

GLDC under anchorage-independent conditions and found that

GLDCtransforms 3T3 cells readily with a rate exceeding that of

LIN28B (Figures 3G and S3I). Upon transplantation into NOD/

SCID Il2rg/ mice, 3T3 cells overexpressingGLDC consistently

formed tumors in 6/6 transplants, and 3T3 cells overexpressingLIN28B formed tumors in 3/6 transplants, whereas 3T3 cells

overexpressing the empty control vector never formed tumors

(Figures S3JS3L).

To test whetherGLDCcan also transform normal primary HLF

and normal primary human bronchial epithelial (NHBE) cells, we

overexpressed GLDC in HLF and NHBE cells (Figures S3M

andS3O). Both HLFand NHBE cells showeda dramatic increase

in cell proliferation upon overexpression of GLDC alone (Fig-

ures 3H3J and S3P). Surprisingly we found that GLDC also

transforms HLF and NHBE cells readily in vitro (Figures 3K

and S3Q). However, perhaps because primary adult HLF and

NHBE cells are not immortalized, GLDC-overexpressing HLF

and NHBE cells do not form tumors upon transplantation (Fig-

ures S3N and S3R). In contrast, CD166 lung tumor cells, whichalso could not form tumors in vivo,could now initiate tumorigen-

esis ata lowfrequency upon overexpression ofGLDC (Figure3L).

Collectively, our results show thatGLDC is an oncogene that is

both necessary and sufficient to promote tumorigenesis.

GLDC Promotes Tumorigenesis through Its Metabolic

Activity

Although GLDC is a metabolic enzyme, it remained unclear

whether GLDC promotes tumorigenesis through a metabolism-

dependent or -independent mechanism. To address this ques-

tion, we engineered a series of four point mutations within or

near the evolutionarily conserved catalytic active site of the

GLDC enzyme to disrupt its metabolic activity (Figure 4A). These

point mutations comprised three nonlethal GLDC mutations

found in human patients with nonketotic hyperglycinemia

(H753P, P769L, G771R; Figures S4A and S4B) and one mutation

K754A that is predicted to abrogate the covalent bond with the

critical pyridoxal-50-phosphate cofactor (Nakai et al., 2005;

Kure et al., 2006). When we overexpressed these four GLDC

mutants in 3T3 cells, none of them could lead to tumorigenesis

in vivo, whereas wild-type GLDC could, even though all of

them were expressed at high levels similar to that in transformed

A549 cells (Figure 4B). Thus the metabolic activity of GLDC is

required for its tumorigenic function.

In addition, the upregulation of many other upstream enzymes

in the glycine/serine pathway in lung TICs further supports the

idea that metabolic activity in the glycine/serine pathway is re-sponsible for promoting tumorigenesis (Figure 2E). To test this

idea, we also overexpressed PSAT1, PSPH, SHMT1, SHMT2,

and GCAT in 3T3 cells and transplanted them in vivo to test

for cellular transformation and tumorigenesis (Figure 4C). By

2 months, we found that three other glycine/serine enzymes

PSAT1, PSPH, and SHMT2could also transform 3T3 cells to

form tumors in vivo (Figure 4D). Interestingly, we noted that

PSAT1, PSPH, and SHMT2 overexpression only led to a slight

upregulation of GLDC protein (Figure 4E), suggesting that their

tumorigenic activity is due to increased glycine/serine metabo-

lism, rather than indirect upregulation ofGLDC. These findings

indicate that increasedmetabolism in the glycine/serinepathway



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Tumor spheres

120,000 TS-Ctrl-sh

A B C

h h h h

HLF

Time after release in serum

hh

8hc h 4

h

8h

A549

Time after release in serum

h0.8

1

s(g)

Ctrl-sh

GD-sh

0

20,000

40,000

60,000

80,000

100,000

0 2 4

Numberofcells

TS-28B-shTS-GD-sh

1

2

4

1

FOS

CDK1

GLDC

0

2 4c

HSP90

CDK1

HSP90

GLDC

8

244

0

0.2

0.4

0.6

Massofxenografttumou

r 28B-sh

*

**

F

100

125

nies/

3T3

3T3-GD3T3-28B

E

3T3

3T3

D

MCF10A

20

30

express

ion

Days

G

60

70

80

5,0

00

3T3

3T3-GD3T3-28B

0

25

50

75

No.ofadherentcolon

5003T3

** **3T3-GD

3T3-28B

0

10

RelativeGLDCe

0

10

20

30

40

50

60

No.ofagarcolonies/5

3T3

** **

60,000

80,000

100,000

erofcells

HLF-GD

HLF-28B

HLF

H

25

30

35

nies/5,0

00

HLF

HLF-GD

HLF-28B

** **

KJ

100

120

140

colonies/

LF

HLF

HLF-GD

HLF-28B****

HLF

HLF

HL

I

0

20,000

40,000

0 3 6

Numbe

Days 0

5

10

15

20

No.ofagarcolon

HLF

0

20

40

60

80

No.ofadherent

1,0

00HL

Xenograft

LF-GD

HLF-28B

Group No. cells /

injection

No. tumors/

No. injections

CD166-200,000 0/12

100,000 0/12

CD166- GD200,000 4/12

100,000 1/12

L

g

,

Figure 3. GLDC and LIN28B Are Necessary and Sufficient for Malignant Growth

(A) Proliferation curve of tumor sphere (TS) cells with shRNA knockdown of either GLDC (GD-sh) or LIN28B (28B-sh).

(B) Quantitative mass analysis of xenograft tumors formed 30 days after transplanting 100,000 tumor sphere cells with either GLDC knockdown (GD-sh) or

LIN28B knockdown (28B-sh).

(C) Western blot analysis of endogenous GLDC during the cell cycle in synchronized normal human HLFs and transformed A549 cells. HLF or A549 cells were

serum-starved for 72 hr followed by release into serum-containing medium with samples collected at indicated time points. Expression of GLDC, FOS (early



7/14

due to GLDC or other glycine/serine enzymes can exert a potent

tumorigenic effect.

GLDC Regulates Glycine Metabolism, with Effects on

Glycolysis and Pyrimidines

Given that GLDC promotes tumorigenesis through a metabo-

lism-dependent mechanism, we performed metabolomic anal-

ysis to gain deeper mechanistic insights into the GLDC-driven

metabolism changes that lead to tumorigenesis. We used liquid

chromatography-mass spectrometry (LC-MS) to perform me-

tabolomics profiling of HLF cells and 3T3 cells overexpressing

GLDC, as well as A549 lung adenocarcinoma cells with retroviral

knockdown of GLDC, relative to empty vector controls. We

found that glycine-related metabolites, glycolysis intermediates,

serum response), andCDK1 (E2Ftarget) weretested. Normal growing, unsynchronized cells (Cyc)were usedas a control. HSP90was used as a loading control.

CDK1, cyclin-dependent kinase 1; HSP90, heat shock protein 90.

(D) Expression of endogenous GLDC in MCF10A cells transformed by oncogenic KRASG12D

, PIK3CAE545K

, MYC, or MYCT58A

, by qRT-PCR.

(E) Colony formation assay in adherent conditions by seeding 500 3T3 cells overexpressing either GLDC (3T3-GD) or LIN28B (3T3-28B).

(F) Quantitative analysis of colony formation efficiency under adherent conditions as shown in (E).

(G) Quantitative analysis of soft agar colony formation by 5,000 3T3 cells overexpressing either GLDC (3T3-GD) or LIN28B (3T3-28B). Colonies were stained with

INT on day 28.

(H) Proliferation curve of HLF cells overexpressing GLDC (HLF-GD), LIN28B (HLF-28B), or the empty vector (HLF).

(I) Colony formation assay in adherent conditions seeding 1,000 HLF cells overexpressing GLDC (HLF-GD), LIN28B (HLF-28B), or the empty vector (HLF).

(J) Quantitative analysis of colony formation efficiency under adherent conditions as shown in (I).

(K) Quantitative analysis of soft agar colony formation by 5,000 HLF cells overexpressing either GLDC (HLF-GD), LIN28B (HLF-28B), or the empty vector (HLF).

(L)Tumorformationby CD166 lung tumor cells 3 months after overexpression of GLDC. CD166 tumor cells from xenografts were sortedby FACS andinfected

with retrovirus expressing either the empty vector (CD166) or GLDC (CD166 GD), followed by transplantation into mice 24 hr after infection (n = 12 for each

group).

In all panels, error bars represent SEM. *p < 0.05, **p < 0.01. See also Figure S3.

BA

WT

H

753P

K

754A

P

769L

G

771R

V

ector

A

549

8/

8

0/

8

0/

8

0/

8

0/

8

0/

8

8/

8

No. tumors/

No. injections

D E

Group No. tumors/

No. injections

3T3-GLDC 6/6

3T3-PSAT1 3/6

3T3-PSPH 2/6

3T3-SHMT2

1/63T3-SHMT1 0/6

3T3-GCAT 0/6

3T3 0/6

1

10

100

1,000

10,000

100,000

RelativeRN

Aexpressionlevel

C

-Actin

GLDC

GLDC-GFP

R

Figure 4. GLDC Promotes Tumorigenesis through Its Metabolic Activity

(A) Crystal structure of T. thermophilus GLDC near the catalytic active site. The labeled bacterial residues H703b, K704b (purple), P729b, and G731b are

homologous to H753, K754, P769, and G711 of human GLDC. Residues implicated in human nonketotic hyperglycinemia are shown (red). PLP, pyridoxal-5 0-

phosphate cofactor (green).

(B) GLDC protein expression and tumor formation efficiency of 3T3 cells overexpressing wild-type or mutant GLDC. Four point mutations were tested: H753P,

K754A, P769L, and G771R. Incidence of tumor formation was determined 2 months after injection with 1.5 3 106 cells per mouse (n = 8). A549 cells served as

a positive control. WT, wild-type.

(C) Geneexpression in 3T3 cells overexpressing GLDC, PSPH, PSAT1, GCAT, SHMT1, and SHMT2, relativeto 3T3cells with theempty vector,as determined by

qRT-PCR.

(D)Tumor formationefficiency of 3T3 cells overexpressing GLDC, PSPH,PSAT1, GCAT, SHMT1, SHMT2, or theempty vector. Incidenceof tumor formation was

determined 2 months after injection with 1.5 3 106 cells per mouse (n = 6).

(E) GLDC protein expression of 3T3 cells overexpressingGLDC, PSPH, PSAT1, GCAT, SHMT1, andSHMT2.A549 cells served as a positive control.b-actin was

used as a loading control.

In all panels, error bars represent SEM. See also Figure S4.



8/14

and many pyrimidines were significantly perturbed by both

GLDC overexpression and knockdown (p < 0.05; Figures

5A5D). For glycine-related metabolites, we found that sarcosine

(N-methylglycine) levels increased significantly upon GLDC

overexpression and dropped significantly upon GLDC knock-down, indicating that GLDC promotes sarcosine synthesis or

accumulation (Figure 5A). Consistent with this observation,

betaine aldehyde in the betaine-sarcosine-glycine pathway for

glycine synthesis also showed the same pattern of changes (Fig-

ure 5A). Glycine levels decrease withGLDCoverexpression and

increase withGLDCknockdown, in agreement with the fact that

GLDC breaks down glycine irreversibly. In contrast, serine levels

increase with GLDC overexpression and decrease with GLDC

knockdown, suggesting that GLDC promotes serine synthesis

or uptake (Figure 5A).

Surprisingly,GLDCperturbation also led to dramatic changes

in glycolysis and other amino acids (Figures 5B, 5C, andS5A).

Our data suggest that GLDC promotes glycolysis, leading

to the increased synthesis or accumulation of glucose-1-phosphate, phosphoenolpyruvate, pyruvate, and lactate (Fig-

ures 5B and 5C). In fact many of the upstream glycine/serine

metabolism enzymes that we found upregulated in lung TICs,

such as PSAT1, PSPH, and SHMT1/2, channel glycolytic inter-

mediates into de novo serine and glycine biosynthesis (Fig-

ure 2E), suggesting that GLDC is working in a concerted fashion

with these enzymes to promote the glycolysis-serine-glycine

flux. This is supported by our finding that GLDC does not signif-

icantly promote glycine uptake (Figures S5B and S5C) but

promotes glycolysis instead (Figures 5B and 5C).

Finally, our metabolomics analysis also revealed that GLDC

promotes the synthesis or accumulation of pyrimidines, in-

cluding thymidine, deoxyuridine, thymine, uracil, and cytosine

(Figure 5D). The GLDC-catalyzed reaction converts glycine into

CH2-THF (Kume et al., 1991). CH2-THF contains the methylene

group that fuels de novo thymidine synthesis from deoxyuridine

in concert with pyrimidine biosynthesis and hence nucleotide

synthesis during cell proliferation (Tibbetts and Appling, 2010).

Recent studies suggest that early oncogenesis involves aberrant

activation of cell proliferation, which then leads to a crisis of

nucleotide deficiency and replication stress (Bester et al., 2011).

Our observations on pyrimidine synthesis suggest that upregula-

tion of GLDC could promote cellular transformation by over-

coming this deficiency to progress onward in early oncogenesis.

To test whether any of the metabolite changes induced by

GLDC can mimic GLDCs effects on cancer cells, we analyzed

whether an increased exogenous supply of specific metabolitescould rescue GLDC retroviral knockdown in A549 cells. We

found that 10 mM of sarcosine could significantly rescue the

proliferation defect uponGLDCknockdown, with little effect on

control A549 cells (Figure 5F), indicating that increased sarco-

sine-glycine metabolite flux can rescue the effects of reduced

GLDC enzyme. To further test whether the production of

CH2-THF is necessary for GLDCs effects on proliferation, we

tested whether the antifolate drug methotrexate could specifi-

cally abrogate GLDC-induced proliferation by reducing the tetra-

hydrofolate (THF) cofactor needed to produce CH2-THF for

pyrimidine synthesis. Our results show that low doses of metho-

trexate specifically abrogated GLDC-induced proliferation in 3T3

and HLF cells, with little effect on control 3T3 and HLF cells

(Figure 5E). Furthermore, methotrexate in combination with

GLDC shRNA killed transformed A549 cells much more effec-

tivelythan eitheralone(Figure5E), suggesting that a combination

of antifolates with a GLDC inhibitor could completely shut offglycine catabolism to treat cancer cells more effectively. Using

these metabolic data, we constructed a model of how aberrant

GLDC expression might reprogram glycolysis and glycine

metabolism fluxes in cancer cells to promote cancer cell prolifer-

ation and tumorigenesis (Figure 5G).

Prognostic Significance of Aberrant GLDC Expression

in NSCLC Patients

To assess whether our experimental findings on GLDC are rele-

vant to human lung cancer patients in the clinic, we used tissue

microarray immunohistochemistry to examine the prognostic

significance of GLDC expression, tumor size, tumor grade, and

cancer stage in clinical tumor samples from cohorts of NSCLC

patients (n = 143) (Figures 6A andS6;Table S2). Subdistributionhazard ratio (SHR) analysis showed that patients with high or

grade 3+ GLDC expression have a 3-fold higher risk of lung

cancer mortality compared to patients with low or grade 0

GLDC expression, even when adjusted for cancer stage (SHR =

3.01, 95% confidence interval [CI]: 1.486.10, p = 0.002) (Fig-

ure 6B). Cumulative mortality analysis also showed that high

GLDC expression (grade 3+) is significantly associated with

higher cumulative incidence of mortality across 143 NSCLC

lung cancer patients, even when adjusted for cancer stage

(p = 0.005) (Figure 6C). CD166 expression was not significantly

associated with higher mortality in lung cancer patientswhich

was not unexpected given that only 1 in 5 3 103 CD166+ cells

are tumorigenic (Figures S6AS6C). Indeed, coimmunostaining

of lung tumors revealed that GLDC+ cells mostly form a subset

of CD166+ cells, andthat notall CD166+ cells areGLDC+ (Figures

6D and S6D). LIN28B immunohistochemistry was also not signif-

icantly correlated with lung cancer patient mortality (Figures

S6AS6C), although western blots revealed that lung TICs

express a second LIN28B isoform that is indiscernible from

immunohistochemistry staining,thus renderingour LIN28B stain-

ing results inconclusive (Figure S6E). Our immunohistochemistry

results on clinical tumor samples areconsistent with theidea that

lung TICs constitute the bulk of the tumors in late stages of

malignancy and demonstrate that aberrant activation of GLDC

is significantly associated with human mortality in NSCLC

patientsfurther supporting its role as a metabolic oncogene in

human NSCLC.

Aberrant GLDC Expression in Other Cancers

To check whether aberrant GLDC expression is specific to

NSCLC, we examined a variety of other cancers. Surprisingly

GLDC is also aberrantly upregulated in subsets of primary

tumors from other cancers, especially ovarian and germ cell

tumors (Figure 7A; Table S3). Further analysis of 606 human

cancer cell lines showed that 158 (26.1%) cancer cell lines over-

express GLDC, including lines from ovarian, germ cell, cervical,

lung, lymphoma, prostate, bladder, and colon cancer (Figure 7B;

Table S4). To test whether GLDC is also required for growth by

one of these GLDC-overexpressing cell lines, we knocked



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A B

Glycine-related metabolites Glycolysis

4.00

le)

Cells

2.00

4.00 Cells

arcos ne

2 Betaine aldehyde3 Serine

4 Glycine

-

2 Phosphoenolpyruvate

3 Pyruvate

0.25

0.50

1.00

.

1 2 3

Foldchange(Log2scal

3T3-GD/Ctrl

HLF-GD/Ctrl

A549-GD-sh/Ctrl0.03

0.06

0.13

0.25

0.50

1.00

1 2 3 4

Foldchange(Log2sca

le)

3T3-GD/Ctrl

HLF-GD/Ctrl

A549-GD-sh/Ctrl

2.00

4.00

(Log2scale)

1.5

2.0

2.5

ateOD

3T3-GD

3T3 Pyrimidines

1 Thymidine

2 Deoxyuridine

3 Thymine

4 Cytosine

DC

1.5

2.0

2.5A549

A549-GD-sh

0.02

F

0.25

0.50

.1 2 3 4 5

Foldchang

3T3-GD/Ctrl

HLF-GD/Ctrl

A549-GD-sh/Ctrl0.0

0.5

1.0

0 1 2 4 8 24 Hr

Lacta 5 Uracil

0.0

0.5

1.0

0 1 2 4 8 24 Hr

E

0

20

40

60

80

100

120

Agarcolonynumber 0 uM Sarcosine

10 uM Sarcosine

0

20

40

0 0 .1 0. 2 0. 5 1 2 5 1 0 2 0 0 0 .1 0 .2 0 .5 1 2 5 1 0 20

No.ofadherentcolonies

/200cells

Methotrexate ( M)

3T3

Control GLDC

G

Serine

Sarcosine

Glycolysis

Betaine Pyrimidine

t b li

Phosphoserine

Gl cine CH -THF

Tetrahydrofolate (THF)

Methotrexate0

5

10

15

20

0 0.1 0.2 0.5 1 2 5 10 0 0.1 0.2 0.5 1 2 5 10

No.ofadherentcolonies

/200cells

Methotrexate ( M)

HLF

Control GLDC

GLDCAldehyde metabolism

CO2NH3NADH

0

50

100

0 10 20 50 0 10 20 50 0 10 20 50

No.ofsoftagarcolonies

/1000cells

Methotrexate ( M)

A549

Control GLDC shRNA1 GLDC shRNA2

Figure 5. Metabolomics of Cells upon GLDC Overexpression and Knockdown

(AD) Relative fold change in levels of (A) glycine-related metabolites, (B) glycolysis intermediates, and (D) pyrimidines in 3T3 cells with GLDC overexpression

(3T3-GD/Ctrl), HLF cells with GLDC overexpression (HLF-GD/Ctrl), and A549 cells with GLDC knockdown (A549-GD-sh/Ctrl), as determined by LC-MS

metabolomics. (C) Lactate production by 3T3 cells with GLDC overexpression or A549 cells with GLDC knockdown.

(E) Effects of the antifolate drug methotrexate on colony formation after GLDC overexpression or knockdown. 3T3 and HLF cells overexpressing GLDC were

plated at clonal density and exposed to varying concentrations of methotrexate for 8 days. A549 cells with GLDC knockdown were plated in soft agar at clonal

density and exposed to varying concentrations of methotrexate for 14 days.

(F) Effects of sarcosine on soft agar colony formation after GLDC knockdown in A549 cells. One thousand cells were seeded in soft agar at clonal density and

exposed to 10mM sarcosine for 14 days.

(G) Model of metabolic flux changes induced by GLDC.

In all panels, error bars represent SEM. See also Figure S5.



10/14

A GLDC

1+ 3+2+0

Ad

B

GLDC Intensity SHR (95% CI) P-value

0.005

1 1.28 (0.61 2.71) 0.514

0.8

0.6

1.0C GLDC3+

1+2+

0

tiveincidence

2 1.19 (0.50 2.84) 0.700

3 3.01 (1.48 6.10) 0.002

D

0.2

.

Time to lung cancer death (years)

P = 0.005

n=143

Cumul

0 2 4 6 8 10 12 14

0

CD166

CD166 GLDC DAPIDAPI CD166 GLDC DAPIDAPI

GLDC GLDCCD166

Figure 6. GLDC Is a Prognostic Indicator for Mortality in NSCLC Patients

(A) GLDCimmunohistochemistrystaining in a NSCLCtumor microarray (n = 143).Representative images shown for humanprimarylung adenocarcinomas (AdC)

immunostained with GLDC. Staining intensity grade is indicated in the upper right corner. The boxed regions in the upper images are shown at higher magni-

fication in the lower images. Scale bar, 100 mm.

(B) Subdistribution hazard ratios for each GLDC staining intensity grade, adjusted for American Joint Committee on Cancer (AJCC) staging. CI, confidence

interval.

(C) Cumulative incidence of lung cancer mortality adjusted for AJCC staging, for patients with each GLDC staining intensity grade.

(D) Coimmunofluorescence staining of CD166 (red) and GLDC (green) on primary lung cancer patient tumors, counterstained with DAPI (blue). Representative

cases with coexpression of high levels of CD166 and high levels of GLDC (left panel) and low levels of CD166 and low levels of GLDC (right panel) are shown.

Higher magnification inset is shown in bottom left corner. Scale bar, 50mm.

See alsoFigure S6andTable S2.



11/14

down GLDC in CACO2 cells. Indeed we found that GLDC knock-

down reduced their proliferation and tumorigenic potential upon

transplantation, suggesting thatGLDCmay act as an oncogene

in other cancer cells as well (Figures 7C7E). To examine the

possibility that GLDC is a housekeeping gene for cell prolifera-

tion, we also knocked down GLDC in normal HLFs (Figure S7A).

We found that HLF proliferation was unaffected by retroviral

knockdown of GLDC (Figures S7B and S7C). Furthermore we

observed that GLDC is highly expressed only in a few normal

tissues, including postmitotic liver cells, kidney cells, placenta

cells, and olfactory bulb neurons (Figure S7D). Altogether our

observations in both experimental and clinical settings suggest

that human GLDC is not a housekeeping gene required for cell

proliferation but rather an oncogenic metabolic enzyme aber-

rantly upregulated in NSCLC and possibly several other human

cancers.

DISCUSSION

TIC State in Lung Cancer

Our work sheds new light on the nature of the TIC state and the

role of metabolic reprogramming in tumorigenesis. In this study,

we isolated a subpopulation of TICs from NSCLC patients using

themarker CD166 andshowed that both theoncogenic stem cell

2

3

4

LDCin

sues

A GLDC expression across primary tissue cancers

-4

-3

-2

-1

0

1

Ovary

Germc

ell

Cervix

Brain

Lung

Lymphoma

Prostate

Head&N

eck

Esophagus

Bladder

Colon

Log2

foldchangeof

tumor/norma

ltis

10000B

1

10

100

1000

NormalizedGLDCintensity

250

0.5

1.0

RelativeGLDC

expression

Ctrl-shGD-sh

C

CACO2

600,000

800,000

fcells

Ctrl-sh

GD-sh

28B-sh

D E

Ctrl-sh GD-sh 28B-sh

.

0

200,000

400,000

0 2 4 6

Numbero

Days0.0

0.5

1.0

R

elativeLIN28B

expression

Ctrl-sh28B-sh

Figure 7. GLDC Is Aberrantly Expressed in Other Cancers

(A) Log2-transformed fold changes in GLDC expression of patient tumors versus normal adjacent tissues across different cancers. Data are normalized and

aggregated from 51 GEO datasets containing 84 sets of tumor expression data (tumor versus normal) with 2,020 tumor samples and 671 normal samples. Fold

change cutoff was set at 1.5 by yellow line.

(B) NormalizedGLDCexpression across various cancer cell lines (n = 606). Yellow line indicates the mean value (250) ofGLDCexpression intensities between

patient lung tumors and normal lung tissues.

(C) qRT-PCR forGLDCand LIN28Bin CACO2 colon cancer cells expressing shRNA against GLDC(GD-sh) orLIN28B(28B-sh).

(D) Proliferation curve of CACO2 cells expressing shRNA againstGLDC(GD-sh) orLIN28Bgene (28B-sh) described in (C). Cell numbers were measured on days

2, 4, and 6.

(E) Tumorformation in mice upon injecting2.53 104

CACO2 colon cancer cells withGLDC knockdown (CACO2-GD-sh)or LIN28B knockdown (CACO2-28B-sh).

Mice were assessed by week 13 (n = 6).

In all panels, error bars represent SEM. See also Figure S7andTable S3 and S4.



12/14

factor LIN28B and the glycine metabolism enzyme GLDC drive

the tumorigenicity of lung cancer TICs.

Our data showed that CD166 enriched for TICs in primary

NSCLC, and that CD166 served as an inert surface marker. In

contrast, our results on CD133 are different from the results re-ported by Eramo et al. (2008) even though both studies used

the same CD133 antibody. This is most likely due to differences

in the xenotransplantation assays, which tend to underestimate

the true frequency of TICs. We employed a more sensitive

mouse xenotransplantation assay using NOD/SCID Il2rg/

mice instead of SCID mice, and we directly transplanted primary

tumor cells with Matrigel instead of expanding the tumor cells

in vitro. Previous studies have demonstrated that using a more

sensitive mouse xenotransplantation assay dramatically im-

proves our understanding of TICs (Quintana et al., 2008). Our

present study supports this notion, leading us to CD166 as a

new marker for the lung TIC-containing fraction. In normal phys-

iology, CD166 is expressed predominantly during embryonic

development, including the embryonic upper airway, primitivecardiac cells, and mesenchymal stem cells (Avril-Delplanque

et al., 2005; Murakami et al., 2007; Hennrick et al., 2007; Sabatini

et al., 2005). Expression of CD166 in the embryonic lung is

consistent with our observation that CD166+ lung TICs express

high levels of embryonic lung transcription factors like PEA3

and thetrachealess homologNPAS1, as well as the oncogenic

stem cell factor LIN28B(Liu et al., 2003; Levesque et al., 2007;

Viswanathan et al., 2009). Interestingly, mouse Lin28 is also

expressed in the embryonic lung during normal development

(Yang and Moss, 2003). These observations suggest that the

TIC state in lung cancer is similar to the embryonic lung progen-

itor state in many aspects.

GLDC Is a Metabolic Oncogene

Our results demonstrate that multiple components in the glycine/

serine pathway are also oncogenes. In addition to embryonic

lung factors, lung TICs also express high levels of GLDC,

GCAT, SHMT1/2, PSPH,and PSAT1, suggesting that TICs rely

on glycine/serine metabolism for tumorigenesis. Overexpression

of catalytically active GLDC, as well as PSAT1, PSPH, and

SHMT2, could induce cellular transformation in 3T3 cells to

form tumors, whereas retroviral knockdown of GLDC signifi-

cantly reduces the tumorigenicity of lung cancer cells. We further

observed that GLDC+ cells mostly form a subset of CD166+ cells

in lung tumors.

PSAT1, PSPH, and SHMT1/2 lie upstream of GLDC in

the glycine/serine pathway, diverting glycolytic flux from3-phosphoglycerate through serine to glycine. GLDC is an

oxidoreductase that catalyzes the irreversible rate-limiting step

of glycine catabolism, by breaking down each glycine molecule

in the glycine cleavage system to produce NADH, CO2, NH3, and

CH2-THF (Kume et al., 1991). CH2-THF fuels the one-carbon/

folate metabolism pool, which in turn supplies methylene groups

for biosynthesis (Tibbetts and Appling, 2010). Consistent with

these facts, we found that GLDC regulates many metabolites

in glycolysis and the glycine/serine pathway, leading to specific

changes in pyrimidine synthesis. Pyrimidine derivatives like

thymidine, in turn, are required for nucleotide synthesis in cell

proliferation. Recent studies suggest that early oncogenesis

involves aberrant activation of cell proliferation, which then leads

to a crisis of nucleotide deficiency and replication stress (Bester

et al., 2011)a crisis thatGLDC upregulation could overcome

for continued progression in tumorigenesis. Interestingly, we

found that GLDC also increases the levels of N-methylglycineor sarcosine, an oncometabolite implicated in prostate cancer

(Sreekumar et al., 2009). Furthermore, we observed that GLDC

promotes glycolysis. Combined with our findings on LIN28,

which has been shown to promote glucose uptake and glycol-

ysis (Zhu et al., 2010, 2011), GLDC might be cooperating with

LIN28 as well as PSAT1, PSPH, and SHMT1/2 to divert the

glycolytic flux to glycine and produce CH2-THF. These observa-

tions support the notion that the Warburg effect promotes

biosynthesis for tumorigenesis (Hsu and Sabatini, 2008; Vander

Heiden et al., 2009).

GLDC and Glycine Metabolism Are Relevant to Human

Cancer Patients

From the prognostic perspective, aberrant GLDC expression issignificantly correlated with the survival rates of NSCLC patients.

This is consistent with the model that TIC clones expand to

constitute the bulk of the tumor in advanced stages of malig-

nancy (Boiko et al., 2010). Aberrantly increased GLDC is also

widespread in many other human cancers, including lymphoma,

ovarian, germ cell, cervical, prostate, bladder, and colon cancer,

whereas most normal adult human tissues express very low

levels of GLDC. Our experimental data further suggest that in

cancers that rely on GLDC and glycine metabolism, the highly

toxic antifolate drug methotrexate might be initially effective

because it targets TICs, although our data suggest an even

more effective chemotherapy could be potentially achieved by

combining an antifolate drug with a GLDC inhibitor or by search-

ing for a glycine cleavage complex-specific antifolate drug

much like the search for kinase-specific inhibitors in targeted

cancer therapy.

Our study links a glycine metabolism enzyme to lung cancer

and tumorigenesis. Recently several metabolic enzymes have

been linked to cancer in patients, supporting the status of meta-

bolic reprogramming as a new hallmark of cancer (Hanahan and

Weinberg, 2011). In particular, the pyruvate kinase M2 isoform

PKM2, isocitrate dehydrogenase IDH1/2, and phosphoglycerate

dehydrogenase PHGDH have been implicated in multiple

cancers (Christofk et al., 2008; Parsons et al., 2008; Dang et al.,

2009; Locasale et al., 2011; Possemato et al., 2011). Regardless

of the controversy over the frequency of TICs at different stages

of malignancy, our approach shows that characterizing theunique molecular basis that defines cancer cells with tumori-

genic capacity may nevertheless provide novel drug targets for

advancing cancer therapy.

EXPERIMENTAL PROCEDURES

Tumor Cell Preparation

NSCLC tumors were collected from patients according to protocols approved

by the Ethics Committee of the National University of Singapore. Samples

were washed, dissociated, and incubated in DNase and collagenase/dispase.

After incubation, cell clusters and red blood cells were removed. Then single

cells were resuspended and ready for transplantation. See the Extended

Experimental Proceduresfor more details.



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