The lncRNA H19 mediates breast cancer cell plasticity ......H19 was knocked down in the weakly metastatic TA2-C47 or highly metastaticTA2-C13cellsbeforeinjectioninmice,wedidnotdetecttu-mor

SC I ENCE S I GNAL ING | R E S EARCH ART I C L E

CANCER

1Department of Medicine, College of Medicine and Health, Lishui University, Zhejiang323000, China. 2Division of Drugs and Pharmacology, Ningbo Institute of MedicalSciences, Zhejiang 315020, China. 3Laboratory of Medicine, People’s Hospital of LishuiCity, Lishui 323000, China. 4Department of Laboratory Animal Science, Tianjin Med-ical University, Tianjin 300007, China. 5Institute for Nutritional Sciences, ShanghaiInstitutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.*Corresponding author. Email: [email protected] (W.Z.); [email protected] (D.X.)†These authors contributed equally to this work.

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The lncRNA H19 mediates breast cancer cell plasticityduring EMT and MET plasticity by differentiallysponging miR-200b/c and let-7bWu Zhou,1*† Xiao-lei Ye,2† Jun Xu,1 Ming-Guo Cao,1 Zheng-Yu Fang,1 Ling-Yun Li,3

Guang-Hui Guan,2 Qiong Liu,2 Yue-Hui Qian,4 Dong Xie5*

Metastasis is amultistepprocess bywhich tumor cells disseminate from their primary site and form secondary tumorsat a distant site. The pathophysiological course of metastasis is mediated by the dynamic plasticity of cancer cells,which enables them to shift between epithelial and mesenchymal phenotypes through a transcriptionally regulatedprogram termed epithelial-to-mesenchymal transition (EMT) and its reverse process, mesenchymal-to-epithelialtransition (MET). Using a mouse model of spontaneous metastatic breast cancer, we investigated the molecular me-diators ofmetastatic competencewithin a heterogeneous primary tumor and how these cells thenmanipulated theirepithelial-mesenchymal plasticity during themetastatic process.We isolated cells from theprimarymammary tumor,the circulation, and metastatic lesions in the lung in TA2mice and found that the long noncoding RNA (lncRNA) H19mediated EMT andMET by differentially acting as a sponge for themicroRNAsmiR-200b/c and let-7b. We found thatthis ability enabled H19 to modulate the expression of the microRNA targets Git2 and Cyth3, respectively, whichencode regulators of the RAS superfamily member adenosine 5′-diphosphate (ADP) ribosylation factor (ARF), a gua-nosine triphosphatase (GTPase) that promotes cell migration associated with EMT and disseminating tumor cells.Decreasing the abundance of H19 or manipulating that of members in its axis prevented metastasis from grafts insyngeneicmice. AbundanceofH19, GIT2, andCYTH3 in patient samples further suggests thatH19might beexploitedas a biomarker for metastatic cells within breast tumors and perhaps as a therapeutic target to prevent metastasis.

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INTRODUCTIONMetastasis, which causes more than 90% of cancer-related deaths, is amultistage process during which malignant cells spread from theprimary tumor into distant organs (1). The vast majority of tumorsare carcinoma [meaning (the tumors are) derived from an epithelium].Although these tumors can release large numbers of cancer cells into thecirculation, only a small proportion of these epithelial-derived cellssurvive the migration process to infiltrate distant organs, and evenfewer cells successfully form clinically relevant metastases. Successfulmetastatic cells must acquire the ability to invade the tumor stroma, tointravasate and extravasate the vascular endothelium, and, most im-portantly, to survive and thrive in a new tissue environment (2).

The ability to adopt phenotypic changes, including changes be-tween epithelial and mesenchymal phenotypes, helps carcinoma cellsthrough metastatic progression bottlenecks. Cytokine signals inducethe reactivation of developmental epithelial-to-mesenchymal transition(EMT) programs that are implicated in themetastatic process (3). EMTis proposed to provide cancer cells with several prometastatic traits, in-cluding stemness, motility, and resistance to chemotherapy (4, 5). Oneargument that has been raised against a role for EMT in cancer progres-sion is that metastatic tumors examined histologically often exhibit anepithelial-like phenotype and resemble the primary tumor (6–8).Increasing evidence that the reverse process, mesenchymal-to-epithelialtransition (MET), is vital for the successful metastatic colonization of asecondary organ has been published recently (9–12). These studies

support a reversible EMT-METmodel formetastatic tumor cells, whichundergo EMT to intravasate into blood capillaries at the primary tumorsite and to extravasate into the distant organ but then revert to an ep-ithelial phenotype to grow in the secondary site and become a clinicallyrelevant, detectable mass (13–15).

It is increasingly evident that the pathophysiological course of me-tastasis is not dependent solely on epithelial or mesenchymal pheno-types; it is also dependent on the ability of carcinoma cells to flexiblyand dynamically transition between these two states, adapting theirmetastatic behavior to current needs (16–20). However, an unknownquestion in the field is whether cancer cells dynamically switch betweenepithelial and mesenchymal phenotypes as a result of gradual accumu-lation of changes that confer an advantage on the cell, allowing it tothrive under different conditions, or whether metastatic “seed cells” al-ready exist from the onset of primary tumor formation. The classicalview of tumor progression, based on the clonal evolutionary theory ofcancer (21), postulated that metastatic ability is conferred by rare ran-dom mutations in primary tumor cells that then become clonally ex-panded after selection at secondary organ sites (22). This conclusion,based on microarray analysis, suggested that clonal heterogeneitywithin primary tumors endows these different cells with distinctmeta-static capabilities (23–25). Thus, metastatic competence can emergewith selection among preexisting heterogeneous cancer cells in a pop-ulationwithout the need for newmutations (23, 26, 27). Given that theinherent heterogeneous subpopulations within primary tumors are asource for the selection ofmetastatic cancer cells, we sought to identifythe innate characteristics of metastatic cancer cells within the primarytumor to determine whether metastatic competence requires the abil-ity to flexibly shift between EMT and MET.

Thus, we investigated the intrinsic properties of metastatic cancercells using a set of otherwise isogenic tumor cell populations that areable to complete distinct steps of metastasis when implanted into the

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mammary glands of TA1mice.We identified the long noncoding RNA(lncRNA) H19 as being essential for tumor metastasis and have char-acterized the molecular actions of H19 that may be required for its in-volvement in human breast cancer metastasis.

RESULTSH19 is essential for tumor metastasisTo understand the mechanisms by which primary tumor–derived sub-populations differ in their metastatic capabilities, we performed single-cell cloning using serial dilution to establish a series of cell lines from asinglemousemammary tumor that arose spontaneously without chem-ical stimulus in a wild-type TA2 mouse (28–31). Although these celllines form primary tumors within a month with equivalent kinetics,they differ substantially in their metastatic potential (table S1). The be-havior of these tumor lines reflects their origin from distinct subpopula-tions within the same primary tumor having distinct metastaticpotency.We therefore divided these cell lines into three distinct groups,

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nonmetastatic groupN, weaklymetastaticgroup W, and highly metastatic group H(Fig. 1A). We hypothesized that the de-fined metastatic properties exhibited byeach of these subpopulations result fromalterations in the expression of specificgenes. Accordingly, we compared the geneexpression profiles of these three groups todissect the specific genetic and epigeneticchanges associated with their respectivemetastatic abilities.Wecompared the tran-scription profile of these tumor cell linespairwise and assigned differentially ex-pressed genes (changes greater than 2.5-fold) to class X (group W versus groupN), class Y (group H versus group W), andclass Z (group H versus group N) (Fig.1A). Class X comprised 27 up-regulatedand 14 down-regulated genes, class Ycomprised 23 up-regulated and 19 down-regulated genes, and class Z comprised 29up-regulated and 25 down-regulated genes(data file S1).

Among the identified genes, H19 tran-script stood out as an attractive candidatebecause of its high expression in highlymetastatic andweaklymetastatic cell lines,but not in nonmetastatic cell lines (datafile S1). H19 is a maternally imprintedoncofetal gene that does not code for aprotein but transcribes an lncRNA.Quan-titative real time PCR (qRT-PCR) analysisconfirmed thatH19 transcript was expressed5- to 15-fold higher in classW andH tumorcells compared to nonmetastatic class N cells(Fig. 1B). Consistently, a previous microar-ray analysis showed that H19 transcriptwas the most strongly up-regulated geneinmetastatic 4T1 cell–derived primary tu-mors compared to nonmetastatic 67NRcell–derived primary tumors (5). 4T1


and 67NR cells, together with 168FARN and 4TO7 cells, are fourbreast cancer cell lines derived from a single mammary tumor thatarose spontaneously in a wild-type BALB/c mouse. Our quantita-tive real-time PCR showed that H19 transcript is also expressed in168FARN, 4TO7, and 4T1 cells, but not in 67NR cells or in mousemammary epithelial line Scp2 cells (Fig. 1C). These observationsled us to pursue H19 as an attractive candidate for involvement inmetastasis.

To determine whether H19 plays a causal role in tumor metastasis,we testedwhether inhibition ofH19 expression affectsmetastatic ability.To do so, we used a lentiviral shRNA system to stably knock downH19abundance in highly metastatic clone 13 (TA2-C13) and weakly meta-static clone 47 (TA2-C47) cells. H19 shRNA significantly reduced theexpression ofH19 transcript in TA2-C13 andTA2-C47 cells, comparedwith shRNA control (Fig. 1D). The knocked-down cells were also stablytransfected with a firefly luciferase gene, and 4 weeks after implantationinto the mammary glands of syngeneic TA1 mice, metastasis wasexamined by bioluminescence imaging. In contrast to controls, when

Fig. 1. H19 was essential for cancer metastasis. (A) The classification for pair comparison between cell cloneswith differently metastatic potency (group N, nonmetastatic clones; group W, weakly metastatic clones; group H,highly metastatic clones). (B) qRT-PCR analysis of H19 abundance in 16 heterogeneous subclones isolated from theprimary tumor of one TA2 mice with spontaneous breast cancer and normalized to that in clone 1. (C) qRT-PCRassessment of H19 abundance in metastatic 168FARN, 4TO7, and 4T1 cells and nonmetastatic 67NR cells and nor-malized to that in mouse mammary epithelial Scp2 cells. (D) qRT-PCR assessment of H19 abundance in TA2-C13and TA2-C47 cells infected with lentiviral short hairpin RNA (shRNA) control or H19 shRNA and normalized to U6small nuclear RNA (snRNA). (E) IVIS bioluminescence imaging of mice (n = 3 each) or extracted tissue from syngeneicTA1 mice 4 weeks after orthotopic injection of TA2-C13 and TA2-C47 cells transfected with luciferase and H19shRNA or control shRNA. (F) The circulating tumor cells (CTCs) and metastatic lesions were calculated from synge-neic TA1 mice 4 weeks after orthotopic injection of TA2-C13 and TA2-C47 cells transfected with luciferase and H19shRNA or control shRNA. (G) qRT-PCR assessment of H19 abundance of primary tumors from 48 primary breastcancer (PBC) patients and of metastases from 60 metastatic breast cancer (MBC) patients and normalized to U6snRNA (Sh-Ctr, shRNA control; Sh-H19, H19 shRNA). Data are means ± SD from three independent experiments. **P <0.01, ***P < 0.001).

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H19 was knocked down in the weakly metastatic TA2-C47 or highlymetastatic TA2-C13 cells before injection in mice, we did not detect tu-mor cells in the peripheral blood or secondary organs (Fig. 1, E and F).

To assess human relevance, H19 abundance was investigated inprimary tumors from 48 PBC and 60 MBC patients and in normalbreast tissue from 132 donors (sample descriptions are in Materialsand Methods and table S2). The median H19 expression in MBCsampleswas about 2.5-fold higher than that in PBC andnormal samples(Fig. 1G). Together, these data suggest that the lncRNA H19 may beessential for tumormetastasis and that the lncRNAH19 expression cor-relates with metastasis in human breast cancers.

H19 sponges miR-200b/cBecause the lncRNA H19 was highly expressed in both weakly andhighlymetastatic cancer cells and proved to be critical for tumormetas-tasis, we attempted to determine the specific steps of themetastatic pro-cess to which H19 contributes. To do so, we labeled metastatic murineTA2-C13 cells with green fluorescent protein (GFP) and implantedthem into the mammary fat pads of syngeneic TA1 mice. Cells from



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the primary tumor, those circulating inthe peripheral blood, or those that hadmetastasized to the lung were sorted byfluorescence-activated cell sorting (FACS)and calledC13-PT, C13-PB, andC13-LM,respectively (Fig. 2A). Microarray analy-sis revealed that the ArfGAP [adenosine5′-diphosphate (ADP) ribosylationfactor (ARF) guanosine triphosphatase(GTPase)–activating protein (GAP)] GIT2,which we previously identified as a regu-lator of EMT (32), was expressed only inC13-PT and C13-LM cells (Fig. 2B).Western blots showed that C13-PT andC13-LM cells had a greater abundanceof the epithelial marker E-cadherin andlower abundance of mesenchymal mar-kers N-cadherin and vimentin comparedto C13-PB cells (fig. S1, A and B).Consistent with the mRNA expressionprofiling, Git2 expression was greater inC13-PT and C13-LM cells than in C13-PB cells (fig. S1C). These results suggestthatGIT2 expressionmay be dynamicallyregulated in tumor cells between the pri-mary, disseminated, and metastatic sites.

To explain how GIT2 expressionmight be controlled dynamically duringmetastasis, we considered the involvementof microRNAs (miRNAs). In particular,we hypothesized that GIT2 abundancemight be inhibited by miRNAs in C13-PBcells but that this inhibition was abro-gated in C13-LM cells. We found thatmouse Git2 gene was a predicted targetof miR-200b/c (fig. S1, D and E), whichare central players in the maintenance ofthe epithelial phenotype (33, 34). Abun-dance of miR-200b and miR-200c wasincreased in the highly metastatic TA2-C13


cells, aswell asC13-PT,C13-PB, andC13-LMcells, compared toweaklymetastatic TA2-C47 cells and nonmetastatic clone 7 (TA2-C7) cells,which all share the same primary tumor origin (Fig. 2, C and D).WhenmiR-200b andmiR-200cwere inhibited by targeted antagomirs (Fig. 2,E and F), the abundance of GIT2 protein in C13-PT and C13-LM cellswas unaffected, but that in C13-PB cells was significantly increased(Fig. 2, G and H). This supports the idea that Git2 may be a targetof miR-200b/c, but that the functional effects of this targeting aresomehow cell-specific (or, rather, context-specific).

To explore how Git2 transcripts are not down-regulated by miR-200b/c in C13-PT and C13-LM cells, we turned to lncRNAs. A growingbody of evidence indicates that lncRNAsmay act as decoys to sequesteror sponge miRNAs and hence modulate miRNA downstream targets(35, 36); thus, we posited that the lncRNA H19 might sponge miR-200b/c to inhibit their functions. The abundance of the lncRNA H19was substantially increased in both highly and weakly metastatic cellscompared with nonmetastatic cells (Fig. 1B). We performed qRT-PCRfor the lncRNA H19, confirming its expression in TA2-C47, TA2-C13,C13-PT,C13-PB, andC13-LMcells (fig. S2A).RNAimmunoprecipitation (RIP)

Fig. 2. Git2 expression was conditionally targeted by miR-200b/c. (A) Schematic outlining the origin of C13-PT,C13-PB, and C13-LM cells. (B) Gene cluster by microarray analysis of C13-PT, C13-PB, and C13-LM cells using thegenes encoding GIT2, CYTH3, E-cadherin, N-cadherin, and vimentin. For each gene, the average signal among all cellsamples was used as the baseline (onefold). Changes from baseline are represented by color intensity, with up-regulationshown as red and down-regulation shown as green. (C and D) qRT-PCR analysis of miR-200b (C) and miR-200c (D)expression in nonmetastatic TA2-C7 cells, weakly metastatic TA2-C47 cells, and highly metastatic TA2-C13 cells andin TA2-C13–derived cells and normalized to U6 snRNA. (E and F) qRT-PCR assessment of miR-200b (E) or miR-200c (F)abundance in C13-PT, C13-PB, or C13-LM cells transfected with the antagomirs of miR-200b, miR-200c, or their antagomircontrols and normalized to U6 snRNA. (G and H) Western blotting analysis of GIT2 in C13-PT, C13-PB, or C13-LM cellstransfected with the antagomirs of miR-200b (G), miR-200c (H), or their antagomir controls. Densitometry was nor-malized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). E-cad, E-cadherin; Vim, vimentin; PT,primary tumor; PB, peripheral blood; LM, lung metastasis. Data are means ± SD from three independentexperiments. **P < 0.01, ***P < 0.001.

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assays using antibodies against mouse Ago2 (Fig. 3A), which is a corecomponent of the RNA-induced silencing complex (RISC) (37), furthersupported the sponging hypothesis. Ago2 antibody precipitated Ago2protein-RNA complexes from C13-PT, C13-PB, and C13-LM cell ly-sates (fig. S2B), and we found that endogenous H19 was preferentiallyenriched in Ago2 RIPs compared to control immunoglobulin G (IgG)antibody RIPs (Fig. 3B). Moreover, Ago2 RIP samples from C13-PTand C13-LM cells were significantly enriched for endogenous miR-200b/c compared to those from C13-PB cells (Fig. 3C), from whichwe infer that H19 and miR-200b/c were in the same Ago2 complexin C13-PT and C13-LM cells. Additionally, bioinformatics tools (38)revealed potential binding sites in mouse H19 for mouse miR-200band miR-200c: miR-200b/c shared 14-mer conserved H19 target se-quences (340–353), wherein two predicted miR-200b sites start at nu-cleotide position 339 and three predicted miR-200c sites start atnucleotide position 338 (data files S2 and S3).

To further explore whetherH19might act as a “sponge” to sequestermiR-200b/c, we transfected TA2-C7 cells, which have a low abundanceof endogenous H19 (Fig. 1B), with the plasmids psiCHECK2-miR-200b/c 4× (encoding miR-200b/c binding sites; the “sensor” in this ex-periment) and various amounts of pH19 (which expresses full-lengthmouse H19; the “sponge” in this experiment). The relative luciferaseactivity increased in response to pH19 in a dose-dependent manner(Fig. 3D), suggesting that ectopically expressed H19 specifically seques-tered endogenousmiR-200b/c, thereby preventing it from inhibiting lu-ciferase expression. Expression of a mutant H19 (pH19mut1), in whichpredictedmiR-200b/c interaction sites weremutated, did not inhibit lu-ciferase expression (Fig. 3D), confirming that miR-200b/c binding sitesare required for this effect. Luciferase reporter assays in which a lucif-


erase reporter of the 3′ untranslated region (3′UTR) of Git2 (Git2pMirTarget) was cotransfected into TA2-C7 cells with miR-200b/cmimics or their controls. TA2-C7 cells cotransfected with miR-200b/cmimics significantly decreased the activity of luciferase constructscarrying the sequence of Git2 3′UTR (fig. S5A). The effects of H19on the Git2 luciferase reporter were also analyzed with full-lengthmouseH19 (pH19) and itsmutant derivative (pH19mut1). The relativeactivity of luciferase was rescued in pH19 cotransfected cells but not inpH19mut1 cotransfected cells (fig. S5A). Moreover, we knocked downthe abundance ofH19with siRNA (fig. S2, C andD) andmonitored theeffect on the abundance of miR-200b/c and GIT2 abundance. H19 hadno effect on the abundance of miR-200b/c (fig. S5B) but reduced theabundance of GIT2 in C13-PT and C13-LM cells (Fig. 3E). However,GIT2 abundance in C13-PB cells was not rescued because they werewithmiR-200b/c antagomirs (Figs. 2, G andH, and 3F). Together, thesedata suggest that H19 physically associates withmiR-200b/c to functionas a competing endogenous RNA (ceRNA) for miR-200b/c in C13-PTand C13-LM cells.

H19 sponges let-7bAs stated, the data thus far suggested that H19 acts as a ceRNA orsponge for miR-200b/c miRNA in tumor cells within the primary tu-mor and in lungmetastases, but somehow, miR-200b/c remained func-tional in targetingGit2 in tumor cells in the circulation. GIT2 belongs tothe family of GAPs for the small GTPase ARF, called ArfGAPs (39, 40).ARF proteins, which regulate vesicular trafficking and actin remodeling(and thus are implicated in cell migration), cycle between their activeGTP-bound and inactive guanosine diphosphate (GDP)–bound con-formations. ArfGAPs promote hydrolysis of bound GTP and induce


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binding of GDP to inactivate the ARF protein (41),whereas ARF guanine nucleotide exchange factors(ArfGEFs) release boundGDP and facilitate bindingof GTP to activate the ARF protein (42). Because wefound that H19 inhibits miR-200b/c targeting ofGit2, we examined the functional effects on ARFactivity in C13-PT, C13-PB, and C13-LM cells. Wefound that increased amounts of GTP-bound ARFproteins were precipitated by the immobilizedARF-specific effector GGA3 (Golgi-associated,g adaptin ear containing, ARF-binding protein 3)(43) in C13-PB cells relative to C13-PT or C13-LM cells (Fig. 3F). Because GTP binding to ARF isfacilitated by ArfGEFs, we thus hypothesized thatan ArfGEF is functional in C13-PB cells.

Among 10 known mouse ArfGEFs, only thetranscript for cytohesin 3 (CYTH3; also known asGRP1 or ARNO3) (data file S4) was predicted byTargetScan to be targeted by the miRNA let-7 (fig.S2, E and F), and let-7 is reportedly bound andsequestered (sponged) by H19 (44). CYTH3 proteinwasmore abundant in C13-PB cells than in C13-PTand C13-LM cells (Fig. 3G), as was GTP-boundARF6 (Fig. 3F), but the opposite (less in C13-PBcells) was observed for the abundance of GIT2(fig. S1C). Although the amount of seven membersof the let-7 family was relatively similar in subclonesof the three initial cell groups (nonmetastaticTA2-C7,weakly metastatic TA2-C47, and highly metastaticTA2-C13 and TA2-C55 cells) and in C13-PT,

Fig. 3. H19 sponged miR-200b/c. (A) Schematic outlining the Ago2 RIP strategy to validate endogenousmiRNA:H19 binding. (B) qRT-PCR assessment of H19 in C13-PT, C13-PB, and C13-LM cells that were pulleddown by AGO2 or negative control IgG and normalized to U6 snRNA. (C) qRT-PCR detection of miR-200b/cendogenously associated with H19 in C13-PT, C13-PB, and C13-LM cells that were pulled down by AGO2 ornegative control IgG and normalized to U6 snRNA. (D) The luciferase activity assessment in TA2-C7 cellstransfected with sensor (miR-200b/c 4×, psiCHECK2-miR-200b/c 4×), together with 0, 20, 40, 80, or 160 ngof sponge plasmid pH19 or pH19mut1. (E) Western blotting detection of GIT2 in C13-PT, C13-PB, andC13-LM cells transfected with #2 H19 small interfering RNA (siRNA) or siRNA control and normalized toGAPDH. (F) Western blotting detection of guanosine 5′-triphosphate (GTP)–bound ARF6 that were pulleddown by glutathione S-transferase (GST)–GGA3–protein binding domain (PBD) beads in C13-PB, C13-PT,and C13-LM cells and normalized to total ARF6. (G) Endogenous CYTH3 in C13-PT, C13-PB, and C13-LM cellswas detected byWestern blotting andquantified relative toGAPDHas a loading control (siCtr, siRNA control;siH19, H19 siRNA). Data are means ± SD from three independent experiments. **P < 0.01, ***P < 0.001.

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C13-PB, and C13-LM cells (fig. S3A), selectively decreasing the amountof let-7bwith an antagomir increased the abundance of CYTH3 inC13-PT and C13-LM cells but not in C13-PB cells (Fig. 4A and fig. S3, B andC), suggesting that CYTH3 may be a target of let-7b in C13-PT andC13-LM cells. In addition, bioinformatics tools (38) revealed potentialbinding sites formaturemouse let-7b inmouseH19 (data file S3). Ago2RIP experiments showed that endogenous let-7b and H19 were bothpulled down in the Ago2 complex in C13-PB cells, but not in C13-PT and C13-LM cells (Figs. 3B and 4B). Furthermore, luciferase report-er assays demonstrated that the relative luciferase activity of TA2-C7cells transfected with psiCHECK2-let-7b 4× (the sensor) increased inresponse to pH19 (the sponge) in a dose-dependent manner, whereasthe sensor was unaffected by pH19mut2, in which predicted let-7b in-teraction sites were mutated (Fig. 4C). Furthermore, H19 knockdownwith siRNA had no significant effect on the abundance of let-7b (fig.S5C) or CYTH3 in C13-PT or C13-LM cells but decreased the abun-dance of CYTH3 in C13-PB cells (Fig. 4D), indicating that H19 com-


peted with let-7b’s ability to bind and down-regulate CYTH3transcripts. Together, these data demonstrated that the lncRNA H19sponged miR-200b/c in C13-PT and C13-LM cells but sponged let-7bin C13-PB cells, mediating the regulation of GIT2 and CYTH3 abun-dance, respectively.

Context-specific sponging by H19 mediatesepithelial-mesenchymal plasticityGiven the relationship between GIT2, CYTH3, and EMT (32, 40), wenext investigated the role of GIT2 and CYTH3 on ARF activity and ep-ithelial or mesenchymal state in C13-PT, C13-PB, and C13-LM cells.Our data show that the knocking down of GIT2 increased the amountof active, GTP-bound ARF6 (fig. S4A) and reduced the abundance ofthe epithelial marker E-cadherin (fig. S4B) in C13-PT and C13-LMcells, whereas CYTH3 knockdown decreased the amount of activeARF6 (fig. S4C) and increased E-cadherin abundance (fig. S4D) inC13-PB cells.



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We then examined the functional ef-fect of potentially selective sponging ofmiR-200b/c and let-7b by the lncRNAH19. The miR-200b/c antagomir in-creased E-cadherin abundance in C13-PBcells (fig. S4E),whereas the let-7b antagomirreduced E-cadherin abundance in C13-PTand C13-LM cells (fig. S4F). Most impor-tantly, forced expression of a miR-200b/c–resistant mutant of the Git2 constructdecreased the amount of active ARF6and increased the amount of E-cadherinin C13-PB cells (fig. S5, D and E), indicat-ing that the endogenousGit2 transcript inC13-PB cells was targeted by miR-200b/cand thus could not inhibit translation ofArf6. On the other hand, expressing let-7b–resistant CYTH3 mutants increasedthe amount of active ARF6 and decreasedthe amount of E-cadherin in C13-PT andC13-LM cells (fig. S5, F and G). Fur-thermore, knocking down H19 reducedE-cadherin and increased vimentin abun-dance inC13-PTandC13-LMcells,whereasthe opposite was observed inC13-PB cells(fig. S4, G and H). These data reveal thatGIT2 is essential for maintaining the epi-thelial state, whereas CYTH3 is critical forinducing a mesenchymal phenotype,and that the abundanceof each—andhenceEMT/METdynamics (at least inmetastaticmurine TA2-C13 breast cancer cells)—is regulated by context-specific spong-ing of miR-200b/c and let-7b by thelncRNA H19.

CYTH3 and GIT2 are involved inmetastatic initiation and metastaticcolonization, respectivelyTo relate these findings to cancer metas-tasis, we tested the involvement ofCYTH3and GIT2 in tumorigenic and metastatic

Fig. 4. H19 sponged let-7b. (A) Western blotting detection of CYTH3 protein expression of C13-PT, C13-PB, andC13-LM cells transfected with let-7b antagomir or antagomir control and normalized to GAPDH. (B) qRT-PCR de-tection of let-7b endogenously associated with H19 in C13-PT, C13-PB, and C13-LM cells that were pulled down byAGO2 or negative control IgG and normalized to U6 snRNA. (C) The luciferase activity assessment in TA2-C7 cellstransfected with sensor (let-7b 4×, psiCHECK2-let-7b 4×), together with 0, 20, 40, 80, or 160 ng of sponge plasmidpH19 or pH19mut2. (D) Western blotting detection of CYTH3 in C13-PT, C13-PB, and C13-LM cells transfected with#2 H19 siRNA or siRNA control and normalized to GAPDH. (E) Number of CTCs in syngeneic TA1 mice 4 weeks afterorthotopic injection of C13-PT, C13-PB, and C13-LM cells infected by CYTH3 shRNA or control shRNA. (F) Bio-luminescence (Biolum), hematoxylin and eosin (H&E) staining, and immunohistochemistry staining with luciferaseantibody (Anti-Luc) of lung metastatic lesions of TA1 mice (n = 3) 4 weeks after orthotopic injection of C13-PT cellsinfected with Git2 shRNA or control shRNA. (G) Quantification of bioluminescence of region of interest (ROI) for lungmetastatic lesions of TA1 mice (n = 3) 4 weeks after orthotopic or tail vein injection of C13-PT cells infected with Git2shRNA or control shRNA. (H) Immunostaining for CTCs in peripheral blood samples from MBC patients. CTCs aredefined as CK+/CD45−/DAPI+ cells with cytokeratin 8 (CK8) (red), CD45 (green), and 4′,6-diamidino-2-phenylindole(DAPI) (blue). Scale bars, 10 mm. NC, negative control; BF, before filtrating; AF, after filtrating; siCtr, siRNA control;siH19, H19 siRNA. Data are means ± SD from three independent experiments. **P < 0.01, ***P < 0.001.

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behaviors, namely, invasion, proliferation, and colony-forming ability.GIT2 knockdown significantly promoted the invasive ability of C13-PT and C13-LM cells, whereas CYTH3 knockdown significantly sup-pressed the invasive ability of C13-PB cells (fig. S4, I to L) in Transwellassays. Furthermore, GIT2 knockdown reduced the colony-formingability and proliferation of C13-PT and C13-LM cells (fig. S4, M, O,andQ),whereasCYTH3knockdown increased the colony-forming abil-ity and proliferation of C13-PB cells (fig. S4, N, P, and R) as assessed bysoft agar and 5-bromo-2′-deoxyuridine (BrdU) incorporation assays.

We then examined the necessity of GIT2 and CYTH3 for successfulmetastasis in two mouse models. C13-PT, C13-PB, and C13-LM cellswere injected into syngeneic TA1 mice either orthotopically into amammary fat pad or into the tail vein. Cells from orthotopically ini-tiated tumors failed to intravasate when CYTH3 was knocked down(Fig. 4E). Cells from either orthotopically initiated tumors or tail veininjections failed to form macrometastasis in the lung when GIT2 wasknocked down (Fig. 4, F and G). These data thus far demonstrate thatCYTH3 promoted metastatic initiation (presumably EMT) but thatGIT2 promoted metastatic colonization (presumably MET).

Last, we evaluated the involvement of GIT2 and CYTH3 in humanbreast tumormetastasis by correlating the abundance of eachwith path-ological phenotypes in clinical breast tumor samples. We obtainedprimary breast tumor specimens, paired CTCs, and metastatic samplesfrom 13 MBC patients. CTCs were isolated by an optimized filtrationmethod and verified by immunostaining as CK+/CD45−/DAPI+ cells(Fig. 4H). In agreement with our findings that GIT2 and CYTH3 weredynamically expressed in primary tumor, circulating, and metastaticcancer cells (that is, C13-PT, C13-PB, and C13-LM cells; Fig. 3G andfig. S1C), GIT2 expression was down-regulated and CYTH3 expressionwas up-regulated in CTCs (as assessed by single-cell qRT-PCR) com-pared to each in primary tumors and metastatic nodules from MBCpatients (Fig. 5, A and B). Together, our findings demonstrated thatthe lncRNA H19 critically contributes to metastatic progression bysponging miR-200b/c or let-7b during distinct phases of the metastaticprocess.


DISCUSSIONMetastasis is a complexmultistep process in which cells from a primarytumor spread to distant organs. Cancer cells must exit the primary tu-mor to invade through the surrounding tissue, intravasate into a bloodor lymphatic vessel, be carried to a distant site, extravasate from the ves-sel into a foreign tissue, and proliferate to reestablish a tumor mass inthe new organ environment (45). The vast majority of disseminatedcancer cells fail to survive and proliferate after invading a foreign tissue(46). However, studies of clonal cell lines derived from late-stage humancarcinomas (47) have provided direct evidence that individual cancercells within a tumor differ in their metastatic capability, including cellsthat are not evenmetastatic, confirming the heterogeneity shown in pre-clinical studies with murine (22) and human tumors (48). Still, theintrinsicmechanisms governing the ability of themetastatic subpopulationwithin the heterogeneous primary tumor to successfully metastasize tosecondary organs are incompletely understood.

Here, we reported that the lncRNA H19 differentially spongedmiR-200b/c and let-7b in a cell context–specific manner, therebycontrolling the abundance of the ArfGAPGIT2 or the ArfGEF CYTH3and the reversible shifts between epithelial and mesenchymal states intumor cells (Fig. 5E). These seemingly contradictory functions of H19were essential to successful metastasis and were differentially harnessedby a tumor cell depending on its state within the process of tumormetastasis.

Our results also highlight a central role for ARF6 activity in regulat-ing distinct aspects of metastasis (49–51). GIT2 is an ArfGAP proteinthat reduces ARF6 activity (52). Disseminated tumor cells had reducedGIT2 abundance and increasedGTP-boundARF6 compared to each incells derived from primary tumor or cells from metastatic lesions.CYTH3 is an ArfGEF that increases ARF6 activity (53). Conversely,disseminated tumor cells had greater abundance of CYTH3 and GTP-bound (active) ARF6 than cells from primary tumor or cells frommetastatic lesions. Thus, regulation ofARF6might be the final commonmediator of the regulatory network composed of the lncRNA H19,miR-200b/c and let-7b, and GIT2 and CYTH3.

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The cell lines used in this study with distinct meta-static potencywere derived froma singlemammary tu-mor that arose spontaneously in a TA2 mouse. Thesecells reflect the intrinsic cellular heterogeneity within aprimary tumor and their ability to engage into distinctsteps in themetastatic process. All metastatic and non-metastatic cell lines expressed high amounts of let-7b,whereas only metastatic cells expressed miR-200b/cand nonmetastatic TA2-C7 cells expressed relativelylow amounts ofH19, suggesting thatH19might be afactor for metastasis. The lack of expression of H19in nonmetastatic cellsmay account for their inabilityto metastasize. Although weakly metastatic cellsexpressed H19 and let-7b miRNA, these cells failedto colonize secondary sites because they had nomiR-200b/c. Accordingly, we propose that in highlymetastatic cells, high expression ofH19 results in se-lective sponging of miR-200b/c and let-7b, con-ferring on these cells the ability to accomplish eachof the EMT-MET transitions required for successfulmetastasis, compared to the weakly metastatic ornonmetastatic cell lines (Fig. 5, C and D).

The miR-200 family members, including miR-200c, directly target and repress Zeb1 expression,

Fig. 5. ArfGAP GIT2 and ArfGEF CYTH3 are involved in metastatic initiation or colonization. (A andB) Western blotting for GIT2 (A) and CYTH3 (B) in the primary breast tumor specimens, peripheral bloodsamples, and metastatic samples from the same patient of 13 MBC patients. (C and D) A possible mech-anism explains the sequential metastatic capability of nonmetastatic TA2-C7, weakly metastatic TA2-C47,and highly metastatic TA2-C13 cells. (E) Schemes describing the mechanism of H19 regulating the stepacquirement of the competence to flexibly shift EMT and MET during metastasis.

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and their frequent loss with concomitant increase in ZEB1 abundancepromotes EMTby down-regulating E-cadherin abundance (54). Contro-versially, our data showed that miR-200b/c was highly abundant in cellsfrom the primary tumor, peripheral circulation, and metastatic lesions,whereas the abundance of both E-cadherin and GIT2 were decreased incells from the peripheral circulation. Although theZeb1 transcript was apossible target of miR-200b/c, the Git2 transcript was apparently tar-geted by miR-200b/c (fig. S1, D and E). In addition, we previously re-ported that loss of GIT2 induced the expression ofZeb1 (32). Therefore,we speculate that miR-200b/c might directly targetGit2 instead of Zeb1in disseminated tumor cells and thus decrease the abundance of GIT2,promote the abundance of ZEB1, and induce EMT. Consistently, themiR-200b/c antagomir restored both GIT2 and E-cadherin abundancein disseminated tumor cells. On the other hand, the amount of E-cad-herin was rescued by inhibiting either H19 or miR-200b only in disse-minated tumor cells, suggesting that miR-200b/c works by targetingGit2 in circulating cancer cells due to sponging by H19.

Immunohistochemistry staining showed that E-cadherin was highlyabundant in tumors of mice injected with cells isolated from the circu-lation or from metastatic lesions. Additionally, in these tumors, theabundance of both GIT2 and CYTH3 was similar to that in “parental”cells (those in the original primary tumor), indicating that the cells inthe circulation and metastases can flexibly reverse their phenotypes inresponse to the newmicroenvironment through intrinsic regulation bylncRNA (H19)–mediated selective sponging.

Our results demonstrate that the lncRNA H19 is more abundant inmetastatic cancer cells than in nonmetastatic cancer cells or normalmammary epithelial cells and that the H19 transcript is more abundantin cells in the peripheral blood frompatients withMBC than from thosewith PBC. It remains to be determined howandwhy the lncRNAH19 isinduced in metastatic cancer cells. Nevertheless, our findings suggestthat the lncRNAH19might be a biomarker formetastatic breast cancerand a therapeutic target to prevent metastasis.

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MATERIALS AND METHODSshRNAs, siRNAs, miRNAs, and inhibitorsH19 SMARTvector Lentiviral shRNA, GIT2 SMARTvector LentiviralshRNA, CYTH3 SMARTvector Lentiviral shRNA, and shRNA controlwere purchased from GE Healthcare Dharmacon Inc. (data file S5 forsequence information). Three unique 27-mer siRNAduplexes ofmouseH19-specific siRNAs (#SR402967) were purchased from OriGeneTechnologies (sequence information in data file S5). The mouse let-7bmiRNAmimics hsa-let-7b (stem-loop accession number: MI0000063),miR-200bmiRNAmimicsmmu-miR-200b (stem-loop accession num-ber: MI0000342), and miR-200c miRNA mimics mmu-miR-200c(stem-loop accession number: MI0000650) were purchased fromAmbion/Life Technologies. The mouse let-7b antagomir (#MmiR-AN0004-AM02), miR-200b antagomir (#MmiR-AN0300-SN20),miR-200c antagomir (#MmiR-AN0302-AM02), and control antagomir(#CmiR-AN0001-SN) were purchased from GeneCopoeia.

PlasmidsGFP (#30127), psiCHECK2-let-7 4× (#20930), and luciferase (#17186)plasmids were obtained fromAddgene. H19mouse open reading frameclone (#MC207056),GIT2mouse complementary DNA (cDNA) clone(#MC220430), CYTH3 mouse cDNA clone (#MC209242), and GIT2pMirTarget luciferase reporter (#SC209774) were purchased fromOriGene Technologies.


To make pH19mut1 in which miR200b/c binding sites were mu-tated and pH19mut2 in which let-7b binding sites were mutated,PCR was carried out using pCMV6-Kan/Neo-pH19 vector as a tem-plate with primers (data file S5). The resulting PCR fragment was li-gated to pCMV6-Kan/Neo opened with Hind III and Xho I. To makepsiCHECK2-miR-200b/c 4× and psiCHECK2-let-7b 4×, an annealedoligonucleotide fragment containing copies of miR-200b/c or let-7bbinding sites (data file S5) was inserted into psiCHECK2-let-7 4×opened with Xho I and Not I. To make Git2 constructs with mutantfor miR-200b/c binding sites and CYTH3 constructs with mutant forlet-7b binding sites, PCR was carried out using GIT2 mouse cDNAor CYTH3 mouse cDNA as a template with the primers (data file S5).The resulting PCR fragment was ligated to pCMV6-Kan/Neo (carryingGIT2 or CYTH3 cDNA) opened with BamHI and Nco I to replace thecorresponding miRNA binding sites.

Microarray analysisTotal RNA samples from 16 subclones (groupN, group P, and group F)or C13-PT, C13-PB, and C13-LM cells were isolated using the RNAisolation kit (Qiagen) according to the manufacturer’s protocol. TotalRNA (100 ng) was reverse-transcribed to produce cDNA/mRNAhybrids, whichwere used as a template to create double-stranded cDNAwith a uniqueDNA/RNAheteroduplex at one end. The cDNAwas thenamplified via SPIA (single primer isothermal amplification), whichproduces single-stranded anti-sense DNA. Post-SPIA modificationgenerates sense target cDNA that was fragmented, biotin-labeled, andhybridized toAffymetrixMouseGene 1.0 ST arrays for 18 hours at 45°Cwhile rotating at 60 rpm (Affymetrix). Arrays were then washed anddeveloped using the FS450_0007 fluidics protocol and scanned usingan Affymetrix 3000 7G scanner.

Fluorescence-activated cell sortingTo generate C13-PT and C13-LM cell cultures, primary tumor tissueand lung metastatic nodules from mice orthotopically injected withGFP-labeled TA2-C13 cells were dissected and crushed in RPMI1640 medium and passed through a 100-mm nylon mesh. The filtratewas centrifuged at 50g for 5 min, and the supernatant was collected. Togenerate C13-PB cell cultures, peripheral blood samples from micebearing GFP-labeled TA2-C13 cells were depleted of red blood cellsusing lysis solution [100 mM NH4Cl (pH 7.4)] for not more than5 min. Cells were suspended in a 40% Percoll (GE Healthcare) solutionand overlaid on a 70% Percoll solution. After centrifugation at 800g for20min, the interphase was collected and washed twice with phosphate-buffered saline (PBS). GFP-positive cells were sorted on a FACSAria II(BD Biosciences) cell sorter equipped with a 488-nm laser directly intothe sorting buffer. To culture cells after FACS, collected GFP-positivecells were mixed in 50% medium/50% serum to maximize cell viabilityand plated in one well of a chamber slide and grown until confluent.Cells were passaged and sorted over several cycles to obtain stableGFP-labeled C13-PT, C13-PB, and C13-LM cells.

Ago2 RIP assayTo determine whether the lncRNA H19, miR-200b/c, and let-7b areassociated with the RISC, we performed RNA pull-down assay usingAgo2 antibody to precipitate the RISC and detected H19 or miRNAsfrom the pellet using qRT-PCR. Briefly, C13-PT, C13-PB, and C13-LMcells were rinsed with cold PBS and fixed with 1% formaldehyde for10min. After centrifugation, cell pellets were collected and resuspendedin NP-40 lysis buffer supplemented with 1 mM phenylmethylsulfonyl

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fluoride (PMSF), 1 mM dithiothreitol, 1% protease inhibitor cocktail(Sigma-Aldrich), and RNase inhibitor (200 U/ml) (Life Technologies).The cell lysates were stored at −80°C before use. The supernatant fromcell lysates was collected by high-speed centrifugation. To generateantibody-coated beads, protein G Sepharose 4 Fast Flow bead slurry(GE Healthcare) was rinsed with NT2 buffer (50 mM tris-HCl,150 mM NaCl, 1 mM MgCl2, and 0.5% NP-40) and incubated withantibody against Ago2 (Abcam). Nonimmune mouse IgG (Sigma-Aldrich) was used as negative control. For RIP, the supernatant was in-cubated with the antibody-coated Sepharose beads overnight. Thebeads were rinsed with cold NT2 buffer, followed by incubation withproteinase K (10 mg/ml) (Sigma-Aldrich). The RNAs bound to Ago2antibody were purified with the RNeasy Mini Kit (Qiagen) and wereused for qRT-PCR.

Animal studiesTA1 and TA2mouse strains were bred by the Animal Center of TianjinMedical University (31). Six-week-old female TA1 mice were used fororthotopic mammary fat pad or tail vein injection metastasis model as-says. For bioluminescence tracking of cells in vivo, mice were anesthe-tized with isoflurane (RWD Life Science), intraperitoneally injectedwith D-luciferin (PerkinElmer), and imagedwith an IVIS Lumina II sys-tem (Caliper Life Sciences). Signal quantification was performed withLiving Image Software (PerkinElmer). All mouse experiments wereconducted in accordance with a protocol approved by the InstitutionalAnimal Care and Use Committee at Lishui University.

RNA extraction and qRT-PCRTotal RNA was isolated by TRIzol Reagent (Invitrogen) following themanufacturer’s instructions. After RNA extraction, RNA samples werereverse-transcribed using the High Capacity cDNA Reverse TranscriptionKit (AppliedBiosystems). TheFastStartUniversal SYBRGreenMastermix(Roche) was used for the qRT-PCR. The relative fold changes of candidategeneswere analyzed by using the 2−DDCTmethod. Results were normalizedto U6 snRNA as indicated.

Isolation of CTCs by sizePatient peripheral blood samples (6 ml; anticoagulated with EDTA)were collected after discarding the first 2 ml to avoid potential skin cellcontaminationbynormal epithelial cells. Next, a filtrationmethodwas ap-plied to isolate CTCs using a calibratedmembrane with 8-mm-diameterpores (Millipore). The filtration system consisted of a filtration tubecontaining the membrane (SurExam), a manifold vacuum plate withvalve settings (SurExam), an E-Z 96 vacuum manifold (Omega), anda vacuum pump (Auto Science). Erythrocytes were removed usingred blood cell lysis buffer [154 mM NH4Cl, 10 mM KHCO3, and0.1 mM EDTA (all from Sigma-Aldrich) in deionized water], and thenthe remaining cells were resuspended in PBS (Sigma-Aldrich) containing4% formaldehyde (Sigma-Aldrich) for 5 min before filtration. After thecell suspension was transferred to the filtration tube, the pump valve wasswitched on to reach at least 0.08 MPa; the manifold vacuum plate valvewas then switched on, and filtration began. Isolated CTCs were furtherconfirmed by immunostaining analysis and used for single-cell qRT-PCRwith the Single Cell-to-CT qRT-PCR kit (Thermo Fisher).

Patient eligibilityThis study included 48 PBC patients and 60 MBC patients treated atZhejiang Cancer Hospital from August 2014 to August 2015. Patientsmeeting all of the following requirements were eligible for enrollment:


(i) a diagnosis of invasive breast cancer confirmed by histology, (ii) notreatment before diagnosis, and (iii) provision of voluntary writteninformed consent. Human specimens included primary tumor tissueand adjacent normal tissue (beyond tumor margins) from PBC andMBC patients for analysis of the expression of the lncRNA H19. Ofthe 60 MBC patients, primary tumor, paired peripheral blood samples,and distant metastasis were all collected from 13 patients and assessed(blinded to the patient/tumor status) by a pathologist for the abundanceof GIT2 and CYTH3.

Immunostaining analysisThe peripheral blood ofMBCpatients was collected and fixed with 10%formaldehyde for 10 min, blocked with 10% fetal bovine serum (FBS),and permeabilized with 0.5% Triton X-100 for 20min before immuno-staining. The samples were incubated in the dark with an antibodycocktail containing anti–CK8-PE (1:10), anti–CD45–fluorescein iso-thiocyanate (1:10) and DAPI (1:100) for 30 min at 25°C. The smearswere analyzed under a fluorescence microscope using a 20× objective.CTCs are defined as CK+CD45−DAPI+ cells.

Generation of knockdown cellsGit2 shRNA, Cyth3 shRNA, H19 shRNA, or shRNA control lentiviralsystem vectors were transfected into 293T cells. The lentiviral particleswere packaged and collected following the manufacturer’s guidelineand were mixed with 1 ml of polybrene (10 mg/ml) (#H9268, Sigma-Aldrich) and diluted into 1 ml of Dulbecco’s modified Eagle’s medium(DMEM)without FBS. The dilutedmediumwith lentiviral particleswasadded to cells seeded into 24-well plates. Twenty-four to 48 hours later,the medium was replaced by DMEMwith 10% FBS and 1% penicillin-streptomycin and followed by positive selection using puromycin(2.5 mg/ml) (Invitrogen). The positive clones were further confirmedby Western blotting (for CYTH3 and GIT2) or qRT-PCR (for H19),and the one with the best knockdown efficiency was chosen in ourexperiments.

ARF activity assayARF activity was analyzed byARF6-GTP pull-down assay following themanufacturer’s protocol (Pierce Biotechnology). Briefly, C13-PT, C13-PB,andC13-LMcellswerewashedwithPBS at 4°C andharvested into 500mlof lysis buffer [200mMNaCl, 50mM tris-HCl (pH 7.5), 10mMMgCl2,1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, 5% glycerol, 1 mMPMSF, 9 nM pepstatin, 9 nM antipain, 10 nM leupeptin, and 10 nMchymostatin]. Cell extracts were incubated with 30 mg of GST-GGA3or GST alone immobilized on glutathione-Sepharose for 1 hour at4°C. The pellets were washed three times with lysis buffer. Bound pro-teinswere eluted by 30 ml of elution buffer. GTP-boundARF6was iden-tified by immunoblotting with an ARF6-specific monoclonal antibody.

Colony formation assaysStable GIT2 knockdown C13-PT, C13-PB, and C13-LM cells and theircorresponding control cells (1 × 103 cells each) were seeded into a six-well plate. After culture for 12 days, cells were fixed with 70% ethanoland subsequently stained by 0.2% cresyl violet solution. The imageswere captured, and the colonies were counted by ImageJ software(U.S. National Institutes of Health).

In vitro invasion and BrdU incorporation assaysThe invasive ability of stable GIT2 knockdown C13-PT, C13-PB, andC13-LM cells and their corresponding control cells wasmeasured using

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24-well Transwell plates (8-mm pore size; Corning) as previously de-scribed (55). Cells (5 × 104) were suspended in 200 ml of DMEM andadded into the upper chamberwith a noncoatedmembrane. The cham-ber inserts were coated with Matrigel (BD Biosciences) at 1:7 dilution.For the proliferation assay, cells were seeded into 96-well plates at5000 cells per well for 24 hours and assessed using a BrdU chemi-luminescent cell proliferation enzyme-linked immunosorbent assay(Roche) as previously described (56).

ImmunohistochemistryTumor specimens from lung metastatic nodules of mice bearing C13-PT Git2 shRNA or shRNA control cell tumors were fixed in 4% para-formaldehyde and then embedded in paraffin. Paraffin blocks were cutinto 5-mm-thick sections and stained with H&E or luciferase antibody.

Western blottingCell lysates were lysed in RIPA buffer (Sigma-Aldrich) with CompleteProtease Inhibitor Cocktail (Roche). Cell lysates were stored at −20°Cbefore use. Cellular proteins were separated by SDS–polyacrylamide gelelectrophoresis using a 5% stacking gel and 10% running gel. The mo-lecular weight of candidate proteins was interpolated using Pre-StainedSeeBlue rainbow marker (Invitrogen) loaded in parallel. The mem-branes were probed with the following antibodies: E-cadherin (CellSignaling Technology), vimentin (Santa Cruz Biotechnology), Git2(Santa Cruz Biotechnology), CYTH3 (Zymed Laboratories), andGAPDH (Sigma-Aldrich). Chemiluminescence was induced by Super-SignalWest Dura Substrate (Pierce), and the images were acquired by aLAS-3000 (Fujifilm Life Sciences) and quantified by Multi Gaugesoftware (Fujifilm Life Sciences). Data are presented as means ± SD,based on at least three independent repeats.

Bioinformatics analysisThe miRNA binding sites on H19 were predicted using the web-basedprogram RNAhybrid (38). https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid/. The predicted miRNA let-7 targets were searched fromTargetScan (www.targetscan.org). For the mouse lncRNA H19, thesequence was downloaded from an lncRNA database (www.lncrnadb.org/). For mature mouse miR-200b/c and let-7b miRNAs, sequenceswere downloaded from the miRBase miRNA database (www.mirbase.org/). The sequences and predicted binding results are summarized indata files S2 and S3.

StatisticsStatistical data analysis was performed using SPSS 17.0. Data arepresented as means ± SD, based on at least three independent repeats.Comparisons between two groupswere conducted using two-tailed Stu-dent’s t test, and differences were considered to be statistically signifi-cant when the P value is less than 0.05.

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/10/483/eaak9557/DC1Fig. S1. Git2 transcript is a predicted target of miR-200b/c.Fig. S2. Cyth3 transcript is a predicted target of let-7b.Fig. S3. The functional effect of let-7 assessed with a targeted antagomir.Fig. S4. The role of GIT2 and CYTH3 as analyzed by RIP and in vitro assays.Fig. S5. Luciferase assay results.Table S1. Metastases resulting from orthotopic injection of cells from distinct clones.Table S2. Clinical characteristics of MBC patients.Data file S1. Microarray results.


Data file S2. miRNA and H19 sequences.Data file S3. miRNA target information.Data file S4. ArfGEF target information.Data file S5. siRNA, shRNA, and PCR primer sequences.

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Acknowledgments: We are grateful to F. Miller (Wayne State University) for mouse breastcancer cell lines 67NR, 168FARN, 4TO7, and 4T1. We thank J.-P. Thiery (National University ofSingapore) and R. T. Premont (Duke University) for pre-reviewing our manuscript. Funding:W.Z. was supported by the National Natural Science Foundation of China (81572879). D.X. wassupported by the National Natural Science Foundation of China (3152010390 and 81230058).Author contributions: W.Z. designed and interpreted the experiments and wrote themanuscript. X.-l.Y., J.X., M.G.-C., Z.-Y.F., L.-Y.L., G.-H.G., Q.L., and Y.-H.Q. conducted theexperiments and analyzed the data. D.X. provided input on the manuscript. All authorsprovided comments and critically read the manuscript. Competing interests: The authorsdeclare that they have no competing interests. Data and materials availability: Themicroarray data are deposited at Dryad, doi:10.5061/dryad.t5pv8.

Submitted 20 September 2016Resubmitted 25 January 2017Accepted 25 May 2017Published 13 June 201710.1126/scisignal.aak9557

Citation: W. Zhou, X.-l. Ye, J. Xu, M.-G. Cao, Z.-Y. Fang, L.-Y. Li, G.-H. Guan, Q. Liu, Y.-H. Qian,D. Xie, The lncRNA H19 mediates breast cancer cell plasticity during EMT and MET plasticityby differentially sponging miR-200b/c and let-7b. Sci. Signal. 10, eaak9557 (2017).

10 of 10

http://dx.doi.org/10.5061/dryad.t5pv8


differentially sponging miR-200b/c and let-7bThe lncRNA H19 mediates breast cancer cell plasticity during EMT and MET plasticity by

Dong XieWu Zhou, Xiao-lei Ye, Jun Xu, Ming-Guo Cao, Zheng-Yu Fang, Ling-Yun Li, Guang-Hui Guan, Qiong Liu, Yue-Hui Qian and

DOI: 10.1126/scisignal.aak9557 (483), eaak9557.10Sci. Signal.

tumors. These findings reveal a previously unknown mediator of the EMT/MET phenomenon in metastasis.sequestered let-7b, ultimately activating ARF. H19 abundance was greater in metastatic than in nonmetastatic humanultimately inhibited the migration-related protein ARF. In mesenchymal-like, disseminated cells in the circulation, H19 in tumor cells. In epithelial-like tumor cells in the primary and metastatic sites, H19 sequestered miR-200b/c, whichthe long noncoding RNA (lncRNA) H19 acted as a ''sponge'' for miRNAs to promote an epithelial or mesenchymal switch

found thatet al.the circulation, and metastatic nodules in the lung in a mouse model of metastatic breast cancer, Zhou binding to target transcripts, suppress the abundance of various proteins. By isolating tumor cells from the primary site,(EMT) and its reverse process (MET). These phenotypic shifts are controlled in part by microRNAs (miRNAs), which, by

To metastasize, cancer cells undergo dynamic shifts in phenotype called epithelial-to-mesenchymal transitionA noncoding mediator of metastasis

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The lncRNA H19 mediates breast cancer cell plasticity ......H19 was knocked down in the weakly metastatic TA2-C47 or highly metastaticTA2-C13cellsbeforeinjectioninmice,wedidnotdetecttu-mor