CANCER

Tumor metastasis to lymph nodesrequires YAP-dependentmetabolic adaptationChoong-kun Lee1, Seung-hwan Jeong1,2, Cholsoon Jang3, Hosung Bae1,2,Yoo Hyung Kim1,2, Intae Park1,2, Sang Kyum Kim4, Gou Young Koh1,2*

In cancer patients, metastasis of tumors to sentinel lymph nodes (LNs) predicts diseaseprogression and often guides treatment decisions.The mechanisms underlying tumor LNmetastasis are poorly understood. By using comparative transcriptomics and metabolomicsanalyses of primary and LN-metastatic tumors in mice, we found that LNmetastasis requiresthat tumor cells undergo a metabolic shift toward fatty acid oxidation (FAO).Transcriptionalcoactivator yes-associated protein (YAP) is selectively activated in LN-metastatic tumors,leading to the up-regulation of genes in the FAO signaling pathway. Pharmacological inhibitionof FAO or genetic ablation of YAP suppressed LN metastasis in mice. Several bioactive bileacids accumulated to high levels in the metastatic LNs, and these bile acids activated YAPin tumor cells, likely through the nuclear vitamin D receptor. Inhibition of FAO or YAP maymerit exploration as a potential therapeutic strategy for mitigating tumor metastasis to LNs.

Metastasis to a sentinel lymph node (LN)predicts subsequent metastasis to otherorgans and mortality of cancer patients(1, 2). Most previous studies of tumormetastasis have focused on distant me-

tastasis (3) rather than themechanism bywhichtumor cells survive and grow within the LNs.With mounting evidence that the LNs are afoothold for further tumor dissemination (4–6),elucidating the mechanisms underlying LNme-tastasis is of paramount importance.To investigate these mechanisms, we studied

mouse tumor models. We performed compar-ative transcriptomics analysis of three sequen-tial stages of LN metastasis by using sorted greenfluorescent protein–labeled (GFP+) B16F10 mel-anoma cells from primary tumors and micro-metastatic and macrometastatic tumor-drainingLNs (Fig. 1A and fig. S1, A and B). Heat map andprincipal components analyses of RNA-sequencing(RNA-seq) data revealed global changes in thetranscriptome during tumor progression to LNmetastasis (Fig. 1B and fig. S1, C and D). The topup-regulated gene sets in the LN micro- andmacrometastatic tumors were related to aspectsof lipid biology, such as bile acid metabolism,adipogenesis, fatty acid metabolism, cholesterolhomeostasis, and oxidative phosphorylation (Fig.1, C to F). Pathway analysis of these up-regulatedgenes indicated that the LN-metastatic tumorsstimulated the fatty acid oxidation (FAO) (fig. S1,

E and F) and peroxisome proliferator–activatedreceptor–a signaling pathways (7) (fig. S1, Gand H).Consistent with this finding, our metabolo-

mics analysis revealed higher levels of fatty acidspecies in naïve LNs than in the footpads (theprimary tumor implantation site) of healthycontrol mice (fig. S2A). In addition, the LN-metastatic tumors showed greater accumulationof fatty acids than the primary tumors (Fig. 2A),suggesting that LN-metastatic tumors preferen-tially oxidize fatty acids as fuel in the lipid-richLN niche. To further investigate this metabolicadaptation of LN-metastatic tumors, we imple-mented an in vivo enrichment selection methodto obtain a highly metastatic subpopulation ofB16F10 cells that thrive within the LN paren-chyma. At 3 to 4 weeks after the implantationof B16F10 cells into the footpad, LN-metastaticcells were isolated from the LNs, expanded inculture, and re-inoculated into the footpad (fig.S2B). After three rounds of this in vivo selection,the LN metastasis–prone adapted B16F10 cellsexhibited higher metastatic activity toward LNsthan the parental B16F10 cells (fig. S2C). More-over, the adapted cells preferentially dependedon FAO over glucose or glutamine oxidation asa major pathway for energy production (Fig. 2Band fig. S2D), and they exhibited enhanced FAOcapacity when the growth medium was supple-mented with fatty acid (fig. S2E). The LN-metastasized tumor exhibited an FAO rate aboutfour times that in the primary tumor or lung-metastasized tumor; this increased rate is com-parable to that in brown adipose tissue (fig. S3,A to C, and Fig. 2C).To address the importance of FAO in LN me-

tastasis, we used the mouse B16F10 melanomafootpad implantation model (Fig. 2D). We locallyadministered etomoxir, a clinically approved in-hibitor of FAO, to the anterolateral side of the

animal’s leg. Although etomoxir treatment didnot affect the size of the primary tumor or theanimal’s body weight, it markedly suppressedLN metastasis without affecting the size of thetumor-draining LN (Fig. 2, D to F; fig. S3, Dand E; and fig. S4, A to D). It also markedlysuppressed LN metastasis after surgical removalof the primary B16F10 melanoma (fig. S4, E toG). Etomoxir treatment also suppressed LN me-tastasis in two mouse models of breast cancer,the MMTV-PyMT genetic model and the ortho-topic 4T1 model (fig. S4, H to M). Moreover,etomoxir treatment suppressed tumor growthwhen B16F10 cells or 4T1 cells were directlyimplanted into the LNs (fig. S5, A to E). How-ever, systemic etomoxir treatment did not affectblood-borne metastasis to the lungs in the sys-temic B16F10 or 4T1 cell injection model (fig. S5,F to K). These findings indicate that the meta-bolic shift to FAO in tumor cells is requiredfor the cells’ metastatic growth in the tumor-draining LNs.To identify the molecular events that trigger

the metabolic conversion in the LN-metastasizedtumors, we revisited our RNA-seq data (Fig. 1).The knockdown of only one gene among the on-cogenic signaling genes induced in the meta-static tumors (fig. S6A)—the gene encoding thetranscriptional coactivator yes-associated pro-tein (YAP)—significantly reduced FAO in themetastasis-adapted cells (Fig. 3A and fig. S6B).YAP knockdown did not affect glucose or glu-tamine oxidation (Fig. 3B). Notably, the expres-sion of YAP target genes was induced in theLN-metastatic tumors (Fig. 3C and fig. S6C).Moreover, the retrovirus-mediated overexpressionof hyperactive YAP [YAP-5SA (8)] led to a 2.4-foldincrease in the level of FAO in cultured B16F10cells and a 2.3-fold increase in the growth ofB16F10 melanoma in LNs (fig. S6, D and E).These results indicate that YAP activation is akey molecular event that mediates FAO activa-tion in LN-metastatic tumors.Given that nucleus-localized YAP is viewed as

the activated form of YAP (9), we analyzed thelocalization pattern of YAP in the metastaticLNs. In the tumor cells at the invasive front ofLN-metastatic melanoma, YAP was localized pre-dominantly in the nucleus (Fig. 3, D to F and I,top). A similar pattern of YAP localization wasfound in the tumor cells at the invasive front ofLN-metastatic tumors in theMMTV-PyMT breastcancer model (Fig. 3, G, H, and I, bottom). By con-trast, in the primary tumors and lung-metastatictumors of these two cancer models, YAP waslocalized predominantly in the cytoplasm (Fig.3J and fig. S6, F to I). Moreover, the migratingtumor cells in the lymphatic vessels also ex-hibited cytoplasmic localization of YAP (fig. S7,A and B). Thus, YAP activation was limited tothe tumor cells that had successfully arrived atthe LNs.To determine whether the LNmicroenvironment

triggers YAP activation, we directly implantedB16F10 cells into the inguinal LNs. At 3 daysafter implantation, YAP was localized predom-inantly in the nucleus of the melanoma cells at

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1Graduate School of Medical Science and Engineering, KoreaAdvanced Institute of Science and Technology (KAIST),Daejeon 34141, Republic of Korea. 2Center for VascularResearch, Institute for Basic Science (IBS), Daejeon 34141,Republic of Korea. 3Lewis Sigler Institute for IntegrativeGenomics and Department of Chemistry, PrincetonUniversity, Washington Road, Princeton, NJ 08544, USA.4Department of Pathology, Yonsei University College ofMedicine, Seoul 03722, Republic of Korea.*Corresponding author. Email: gykoh@kaist.ac.kr

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Fig. 1. LN-metastatic tumors undergo transcriptomic changes towardincreased lipid metabolism. (A) Immunofluorescence (IF) imagesshowing naïve popliteal LNs (pLNs) and GFP+ tumors in micro- andmacrometastatic pLNs. Insets show gross images of each LN. Scale bars,1 mm. (B) Heat map of the RNA-seq data from primary (PT), micro-metastatic (Micro), and macrometastatic (Macro) tumors (n = 4 samplesfor each group). (C to F) LN-metastatic tumors showing transcriptional

induction of lipid metabolism. Shown are gene set enrichment analyses[(C) and (D)] and heat maps for the top 30 up-regulated genes for fattyacid metabolism [(E) and (F)] in micro- and macrometastatic tumorscompared with primary tumors. Functions related to lipid biology areunderlined in red in (C) and (D). TNFA, tumor necrosis factor a; NFKB,nuclear factor kB; UV, ultraviolet; IL2, interleukin-2; NES, normalizedenrichment score; FDR, false discovery rate.

Fig. 2. EnhancedFAO is required forLN metastasis.(A) Comparison ofin vivo [14C]oleic acidaccumulation betweenprimary tumors (PTs)and LN-metastatictumors (LMTs) by themeasurement of theradioactive [14C]oleicacid count per minute(cpm) per milligramof the tumor tissue inthe B16F10 melanomafootpad implantationmodel (n=6 tumors foreach). (B) Metastasis-adapted cells (fig. S2B)favor FAO in vitro.The oxygen consumption rates (OCRs) are compared between theadapted and parental cells for glucose, glutamine, and FAO pathways(n = 8 samples for glucose and fatty acid; n = 7 samples for glutamine).(C) Comparison of 14CO2 production rates in primary tumors, lung-metastatic tumors (lung-MTs), LN-metastatic tumors, and brown adiposetissue (BAT) measured by ex vivo FAO assay with [14C]palmitic acid (n = 4

samples for each group). (D) Treatment schedule for the FAO inhibitoretomoxir in the B16F10 melanoma footpad implantation model. (E and F)Comparison of the gross appearances of metastatic pLNs and variousmetastasis parameters between PBS- and etomoxir-treated groups (n =12 samples for each group). Scale bar, 5 mm. AU, arbitrary units. **P < 0.01,***P<0.001, and ****P<0.0001. Error bars are presented asmean±SEM.

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the invasive front (fig. S7, C and D). We nextexamined the effect of YAP depletion on LNmetastasis by using a system for the doxycycline-inducible knockdown of YAP (YAP-iKD) (fig. S7E).To ensure that YAP was depleted after metastasis,

we started mice on a doxycycline diet 2 weeksafter the implantation of the melanoma cells(Fig. 3K). YAP depletion strongly suppressedLN metastasis without significantly changingthe size of the primary melanoma or the size

of the tumor-draining LNs (Fig. 3, K to M, andfig. S7, F to H). YAP depletion after the removalof the primary melanoma also markedly sup-pressed metastatic tumor growth in LNs (fig. S8,A to C). YAP depletion delayed the growth of

Lee et al., Science 363, 644–649 (2019) 8 February 2019 3 of 5

Fig. 3. YAP activation is critical for enhanced FAO and tumor LNmetastasis. (A) Comparison of the [14C]palmitic acid oxidation rates inthe metastasis-adapted B16F10 cells transfected with small interferingRNAs (siRNAs) targeting the indicated genes (n = 4 samples for eachgroup). siCtrl, control siRNA; siMYC, siEGFR, siKRAS, and siAKT, siRNAsfor MYC, epidermal growth factor receptor, KRAS, and AKT, respectively.(B) Comparison of oxygen consumption rates (OCRs) in the adapted cellstransfected with control or YAP siRNA (n = 7 samples for each group).(C) Gene set enrichment analysis (left) and heat map (right) of YAP targetgenes for micrometastatic tumors (Micro) and primary tumors (PTs).FDR, false discovery rate. (D to F) IF images showing metastatic pLNs(D) and highly magnified tumor margins [(E) and (F)]. YAP (green) islocalized in the nuclei at the invasive front (arrowheads in b′), but it islocalized in the cytosol in the intratumoral area (a′). Scale bars, 100 mmfor (D) and (E) and 25 mm for (F). PDGFRb is an LN stromal cell marker.DAPI, 4′,6-diamidino-2-phenylindole. (G and H) IF images showing low-

magnification (G) and high-magnification (H) metastatic axillary LNswith nuclear YAP localization at the invasive front (arrowheads in b′′) inthe MMTV-PyMT spontaneous breast cancer mouse. Scale bars, 25 mm.CK8 is a breast cancer cell marker. (I) Quantification of tumor cells withnuclear YAP in both tumor models (n = 13 and 9 samples for B16F10 andMMTV-PyMT, respectively). (J) IF images of primary tumors of implantedB16F10 melanoma and MMTV-PyMT breast cancer showing cytosolic YAP.Scale bars, 25 mm. (K) Schedule depicting inducible knockdown of YAP(YAP-iKD) in the B16F10 melanoma footpad implantation model. Thedoxycycline diet was started at 2 weeks after the implantation of B16F10cells into footpads, and pLNs were harvested 10 days after the doxycyclinediet. (L and M) Gross appearance of metastatic pLNs and comparisonsof metastatic tumor areas and pLN sizes between control (Ctrl-iKD) andYAP-iKD groups (n = 7 samples for each group). Scale bar, 5 mm. AU,arbitrary units. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.Error bars are presented as mean ± SEM.

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B16F10 melanoma and 4T1 breast cancer cellsimplanted directly into the LNs but had nosubstantial effect on blood-borne metastasis tothe lung in the systemic B16F10 or 4T1 cell in-jection model (fig. S8, D to O). Together, thesefindings indicate that YAP activation plays acritical role in efficient LN metastasis.We next sought to identify the mechanism by

which YAP is activated in LN-metastatic tumors.Although hypoxia and cell proliferation are eachassociated with YAP activation (10, 11), we foundno definitive association between either of thesefactors and the nuclear localization of YAP (fig.S9, A and B). Given that nuclear YAP localizationis restricted to the invasive front in the meta-static LNs, we hypothesized that certain signalingligands present in the LN microenvironmentactivate YAP. We found that the abundance of

several bile acid species was markedly elevatedin the LN-metastatic melanoma (Fig. 4A). Thesebile acids were detected exclusively in the LN-metastatic melanoma and not in normal tissues(footpad tissue and naïve LNs); by contrast,cholesterol—the precursor molecule that givesrise to bile acids—was present at similar levelsin all of these tissues (fig. S9C). Moreover, thesystemic lymph of mice bearing LN-metastatictumors contained higher levels of bile acidsthan that of healthy control mice (fig. S9D).RNA-seq data also revealed highly induced genesignatures of bile acid metabolism in the LN-metastatic tumors compared with primary tumors(Fig. 4B and fig. S9E).In addition to their well-established role in

dietary fat digestion, bile acids can act as sig-naling molecules to activate YAP (12–16). Thus,

we examined whether the bile acids elevated inthe LN-metastatic tumors can activate YAP.Treatment with the bile acid taurodeoxycholicacid (TDCA) induced YAP dephosphorylation(activation) in the cultured adapted B16F10cells within 30 min (Fig. 4C and fig. S9, F toH). The subcutaneous administration of TDCApromoted the growth of LN-metastatic mela-noma by ~2.5-fold compared with the controlvehicle, phosphate-buffered saline (PBS) (fig.S9, I to L). Treatment with cholesterol alsodephosphorylated YAP in the cultured adaptedB16F10 cells but with slower kinetics (Fig. 4Dand fig. S10, A and B), and this effect was blockedby the depletion of CYP7A1, a key rate-limitingenzyme for the conversion of cholesterol intobile acids (Fig. 4E and fig. S10, C and D). Wefound that the cultured adapted B16F10 cells

Lee et al., Science 363, 644–649 (2019) 8 February 2019 4 of 5

Fig. 4. LN-metastatic tumors produce bile acids that can activateYAP, and YAP activation is correlated with melanoma LN metastasisand patient survival. (A) Metabolomics analysis revealing a relativeabundance of bile acids in primary tumors and LN-metastatic tumors inthe B16F10 melanoma footpad implantation model and footpad tissue andnaïve LNs of control mice (n = 3 samples for each group). (B) Gene setenrichment analysis (left) and heat map (right) for the genes related to bileacid metabolism in micrometastatic tumors and primary tumors. (C toE) Immunoblot analyses of phosphorylated and total YAP in adaptedB16F10 cells after treatment with the indicated concentrations of TDCA(C) or cholesterol [(D) and (E)]. In (D), methyl-b-cyclodextrin (MbCD) wasused as a solubilizer for cholesterol (see the blots in fig. S10A). In (E),adapted B16F10 cells were transfected with two different (#1 and #2)

CYP7A1 siRNAs and then treated with cholesterol for 60 min. See fig. S9F,S10B, and S10C for quantifications. (F) Images showing YAP in themetastatic sentinel LN from a melanoma patient. YAP is localized mainly innuclei in the invasive front (right) but not in the intratumoral region(left). The brown color of the metastatic melanoma indicates melaninpigments. Scale bars, 100 mm. (G) Kaplan-Meier survival curves ofmelanoma patients with nuclear or cytoplasmic YAP at the invasive front ofLN-metastatic tumors (n = 8 and 13 patients, respectively). P = 0.0037by a log-rank test. CA, cholic acid; DCA, deoxycholic acid; TCA, taurocholicacid; MCA #1 and MCA #2, two different muricholic acid (MCA) isomers;FDR, false discovery rate; pYAP, phosphorylated YAP. *P < 0.05,**P < 0.01, ***P < 0.001, and ****P < 0.0001. Error bars are presentedas mean ± SEM.

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generated several putative bile acids from cho-lesterol in a CYP7A1-dependent manner (fig. S10,E and F). We also observed a marked suppres-sion of tumor growth, together with decreasednuclear YAP localization, in CYP7A1-depletedB16F10 cells compared with control B16F10 cellsin the LNs (fig. S10, G to K). These findingssuggest that the LN-metastatic tumor itselfproduces bile acids that can activate YAP in anautocrine manner and stimulate further growthof the LN-metastatic tumor; however, this hypo-thesis requires further investigation. We identi-fied nuclear vitamin D receptor (VDR) as thebile acid–activated receptor that likely mediatesthe bile acid–induced YAP activation and tumorgrowth in LNs (fig. S11, A to G). On the basisof these collective data, we postulate that LN-metastatic tumors accumulate bile acids thatactivate YAP mainly via VDR, leading to FAOactivation and successful adaptation to the LNmicroenvironment.Lastly, we investigated the LN-metastatic tumors

in 21 patients withmelanoma (table S1). About halfof the metastatic LNs dissected from patients withmelanoma showed a predominantly nuclear YAPlocalization pattern, specifically at the invasivefront (Fig. 4F and fig. S12A). By contrast, pri-mary melanomas from the same patients didnot exhibit nuclear YAP localization (fig. S12B).Notably, nuclear YAP localization in the meta-static LNs of melanoma patients correlated witha reduction in distant metastasis–free survival(Fig. 4G). This pattern was independent ofpatient age or the thickness of the primarylesion (fig. S12C). YAP activation is therefore akey biomarker that distinguishes LN-metastatictumors with a high risk for metastasis to distantorgans in humans.In this study, by using comparative systematic

analysis of LN-metastatic and primary tumors,we discovered that tumors undergo a metabolicshift toward FAO during LN metastasis. Weidentified YAP, potentially activated by accu-mulated bile acids, as a crucial driver for tumorLN metastasis through selective stimulation ofFAO. The metabolic shift of tumors to aerobic

glycolysis (theWarburg effect) is a well-establishedhallmark of cancer (17). However, different tu-mors may rely on distinct metabolic pathways,such as glutaminolysis, branched-chain aminoacid oxidation, and creatine usage, dependingon their microenvironments (18–20). This flexi-ble fuel choice may allow tumor cells to survivein metastatic sites containing different nutrients(21–25). We found that tumor cells undergoingmetastasis to tumor-draining LNs adapt to theLNmicroenvironment through YAP-dependent ad-justment to the FAO pathway, using the plentifulfatty acids within the node microenvironmentas fuel. Through our metabolomics and tran-scriptomics analyses, we unexpectedly identi-fied bile acids as potential molecular triggersof YAP activation in metastatic LNs. Thus, thespecial microenvironment of the LN, which isenriched with fatty acids as a fuel source andwhich has an abundance of bile acids, may simul-taneously facilitate tumor adaptation and meta-bolic conversion (fig. S13).For a variety of mouse and human tumors,

the LNs are a preferred route for distant meta-stasis (5, 6, 26–28). Selective inhibition of LNmetastasis may prevent or reduce the dissem-ination of tumor cells to distant organs. Ourfindings with mouse models suggest that thera-pies targeting FAO and YAP may be one way tosuppress LN metastasis and subsequent distantmetastasis. Whether these findings will translateto cancer patients remains to be investigated.

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ACKNOWLEDGMENTS

We thank S. Y. Choi, T. W. Noh, S. P. Jang, J. H. Song, andD.-S. Lim (KAIST) and Y. Kim (Asan Medical Center) fordiscussions and experimental support; J. Shin and S. Lee (KAIST)for discussions and statistical support; and S. Seo, J. Bae, andH. T. Kim (Institute for Basic Science) for their technicalassistance. Funding: This study was supported by the Institute forBasic Science, funded by the Ministry of Science and ICT, Republicof Korea (IBS-R025-D1 to G.Y.K.). Author contributions: C.-k.L.designed and performed the experiments, analyzed andinterpreted the data, and wrote the manuscript; S.-h.J. contributedto the majority of the in vivo experiments; C.J. supervised andanalyzed metabolism studies and participated in manuscriptpreparation; H.B. contributed to metabolism experiments; Y.H.K.contributed to in vitro experiments; I.P. contributed to in vivoexperiments and participated in manuscript preparation; S.K.K.analyzed human melanoma–metastatic LNs; G.Y.K. supervised theproject, oversaw data analyses and interpretation, and wrote themanuscript. Competing interests: The authors declare nocompeting interests. Data and materials availability: RNA-seqdata have been deposited in the ArrayExpress database atEMBL-EBI under accession number E-MTAB-7621.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/363/6427/644/suppl/DC1Materials and MethodsFigs. S1 to S13Table S1References (29–40)

6 August 2018; resubmitted 20 November 2018Accepted 15 January 201910.1126/science.aav0173

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Tumor metastasis to lymph nodes requires YAP-dependent metabolic adaptation

Young KohChoong-kun Lee, Seung-hwan Jeong, Cholsoon Jang, Hosung Bae, Yoo Hyung Kim, Intae Park, Sang Kyum Kim and Gou

DOI: 10.1126/science.aav0173 (6427), 644-649.363Science 

, this issue p. 644ScienceImportantly, inhibition of fatty acid oxidation or YAP signaling suppressed lymph node metastasis in the mice.occurs through activation of a signaling pathway driven by the yes-associated protein (YAP) transcription factor.tumor cells adapt to the lymph node microenvironment by shifting their metabolism toward fatty acid oxidation. This

found thatet al.mechanisms that allow tumor cells to survive and grow within lymph nodes. Studying mouse models, Lee bloodstream directly, or they can enter a lymph node adjacent to the primary tumor. Little is known about the biological

Metastatic cells can migrate from a primary tumor to distant organs through two routes: They can enter theFueling lymph node metastases

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