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Tumor Biology and Immunology

Glycoprotein nmb Is Exposed on the Surface ofDormant Breast Cancer Cells and Induces StemCell–like PropertiesChen Chen1,2, Yukari Okita1,3, Yukihide Watanabe1, Fumie Abe4, Muhammad Ali Fikry1,Yumu Ichikawa1, Hiroyuki Suzuki1, Akira Shibuya4, and Mitsuyasu Kato1,3

Abstract

Glycoprotein nmb (GPNMB) is a type I transmembraneprotein that contributes to the initiation and malignant pro-gression of breast cancer through induction of epithelial–mesenchymal transition (EMT). Although it is known thatEMT is associated with not only cancer invasion but alsoacquisition of cancer stem cell (CSC) properties, the functionof GPNMB in this acquisition of CSC properties has yetto be elucidated. To address this issue, we utilized a three-dimensional (3D) sphere culture method to examine thecorrelation between GPNMB and CSC properties in breastcancer cells. Three-dimensional sphere cultures inducedhigherexpression of CSC genes and EMT-inducing transcriptionfactor (EMT-TF) genes than the 2D monolayer cultures.Three-dimensional culture also induced cell surface expressionof GPNMBon limited numbers of cells in the spheres, whereasthe 2D cultures did not. Therefore, we isolated cell surface-

GPNMBhigh and -GPNMBlow cells from the spheres. Cell sur-face-GPNMBhigh cells expressed high levels of CSC genes andEMT-TF genes, had significantly higher sphere-forming fre-quencies than the cell surface-GPNMBlow cells, and showed nodetectable levels of proliferation marker genes. Similar resultswere obtained from transplanted breast tumors. Furthermore,wild-type GPNMB, but not mutant GPNMB (YF), which lackstumorigenic activity, induced CSC-like properties in breastepithelial cells. These findings suggest that GPNMB is exposedon the surface of dormant breast cancer cells and its activitycontributes to the acquisition of stem cell–like properties.

Significance: These findings suggest that cell surface expres-sion of GPNMB could serve as a marker and promisingtherapeutic target of breast cancer cells with stem cell-likeproperties. Cancer Res; 78(22); 6424–35. �2018 AACR.

IntroductionBreast cancer is the most common cancer among women

worldwide, which is a heterogeneous disease and can be classifiedinto five molecular subtypes, luminal A, luminal B, HER2-enriched, basal-like, and normal-like, on the basis of the expres-sion of estrogen receptors (ER), progesterone receptors (PR), andHER2 (1). The basal-like subtype is also referred to as triple-negative breast cancer (TNBC) because it is typically negative forER, PR, andHER2. TNBC accounts for 10%–30%of all diagnosedbreast cancers. In general, TNBC is associated with poor prognosisand high lethality. Moreover, the lack of effective molecularlytargeted drugs limits the treatment options for this aggressivedisease. Therefore, to develop novel molecularly targeted thera-

pies for patients with TNBC, furthermolecular characterization ofTNBC is required (2, 3).

Glycoprotein nmb (GPNMB) is a type I transmembrane pro-tein. Abundant expression of GPNMB was observed in glioblas-toma, melanoma, and breast cancer, especially in TNBC, and wasreported as a prognostic factor (4–7). Moreover, the associationbetweenGPNMB- andHER2-positive breast cancers has also beenreported (8). GPNMB is known to have functions in angiogenesis,tumorigenesis, cell migration, and cell invasion and metastasis,and has received much attention as a target molecule for cancertreatment (9–13).

We previously demonstrated that enhanced expression ofGPNMB induces epithelial–mesenchymal transition (EMT) andincreases anchorage-independent sphere formation and invasivetumor growth in vivo through the hemi-immunoreceptor tyro-sine–based activation motif (hemITAM) in the intracellulardomain. Furthermore, knockdown of GPNMB attenuated thetumorigenic abilities of TNBC cell lines (14). EMT is an essentialprocess during embryogenesis, tissue repair, fibrosis, and cancerinvasion and metastasis. In 2008, Mani and colleagues reportedthat the EMT-inducing transcription factors (EMT-TF) SNAIL andTWIST as well as TGFb signaling are associatedwith acquisition ofcancer stem cell (CSC) properties in breast epithelial cells (15).Evidence of CSC properties induced by EMT-TFs was also shownin breast cancer (16, 17). CSCs harbor the potential of self-renewal, differentiation, tumorigenesis, and resistance to drugsor radiation owing to their ability to enter dormancy and theirabundant expressionofdrug exporters (18–20). For these reasons,CSCs are thought to be the root cause of cancer metastasis and

1Department of Experimental Pathology, Graduate School of ComprehensiveHuman Sciences and Faculty of Medicine, University of Tsukuba, Ibaraki, Japan.2Department of General Surgery, Xin Hua Hospital affiliated to Shanghai JiaoTong University School of Medicine, Shanghai, China. 3Division of Cell Dynamics,Transborder Medical Research Center, University of Tsukuba, Ibaraki, Japan.4Laboratory of Immunology, Life Science Center for Survival Dynamics ofTsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki, Japan.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Yukari Okita, University of Tsukuba, 1-1-1 Tennodai,Tsukuba, Ibaraki 305-8575, Japan. Phone/Fax: 81-29-853-3944; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-18-0599

�2018 American Association for Cancer Research.

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relapse. Therefore, targeting EMT-related molecules might be apromising therapeutic target to eradicate CSCs.

In this study, we investigated whether GPNMB, which inducestumorigenesis and EMT in mammary epithelial cells, affectsacquisition of CSC-like properties in breast cancer cells. Ourfindings propose a novel model in which cell surface expressionofGPNMB induces stemcell–like properties throughhemITAM indormant breast cancer cells. In otherwords, cell surface expressionof GPNMB could be a desirable marker and therapeutic target ofbreast cancer cells with stem cell–like properties.

Materials and MethodsCell lines and cell culture

Breast cancer cell lines BT-474, Hs578T, MDA-MD-468, and4T1were obtained fromATCC (14). In the two-dimensional (2D)monolayer cultures, cells were cultured in DMEM (Invitrogen)supplementedwith 10%FBS (Gibco), 100U/mLpenicillinG, and0.1 mg/mL streptomycin sulfate (Wako). In the 3D sphere cul-tures, cells were cultured in DMEM/F12 (1:1) medium (Invitro-gen) with 2% B-27 supplement (Invitrogen), 2 ng/mL bFGF(Wako), 2 ng/mL EGF (Sigma), 100 U/mL penicillin G, and0.1 mg/mL streptomycin sulfate (Wako) in ultralow attachmentculture dishes (Corning) or poly(2-hydroxyethyl methacrylate)(Poly-HEMA; Sigma)-coateddishes.Hs578T cellswere cultured inthe presence of 10 mg/mL insulin in both the 2D and 3D cultureconditions. NMuMG-mock, NMuMG-GPNMB, and NMuMG-GPNMB (YF) cells were established and maintained as describedpreviously (14). Mycoplasma detection was performed using aMycoplasma Detection Set (Takara) for all the cell lines.

RNA interference4T1 cells were transfected with 40 nmol/L of siRNA directed

against GPNMB or control siRNA (Invitrogen) using Lipofecta-mine 3000 (Invitrogen) following themanufacturer's recommen-dations. Stealth siRNAs (Invitrogen) were purchased as follows:siGpnmb #1 (MSS234870) and siGpnmb #2 (MSS294588).Control siRNA was purchased from Invitrogen (Stealth RNAiNegative Universal Control Medium).

Reverse transcription and qRT-PCRTotal RNA was isolated using Isogen II (Nippon Gene). The

NucleoSpin RNAXSKit (Macherey-Nagel) was used for extractionof total RNA from small samples after cell sorting. Reversetranscription was performed with High Capacity RNA-to-cDNAMasterMix (Applied Biosystems).mRNA levels weremeasured byqPCR with gene-specific primers (Supplementary Table S1) usingSYBR Green I qPCR Master Mix (Applied Biosystems) on the ABI7500 Fast Sequence Detection System. All samples were run intriplicate in each experiment.

Western blot analysisCells were suspended in 62.5 mmol/L Tris-HCl (pH 6.8) and

solubilized in SDS sample buffer [10% glycerol, 5% 2-mercap-toethanol, 2%SDS, and62.5mmol/L Tris-HCl (pH6.8)].Westernblot analysis was performed as described previously (14).

Immunofluorescence and IHC stainingImmunofluorescence and IHC staining methods used were

described previously (14, 21). Anti-GPNMB antibody (AF2550;R&D Systems) was used as the primary antibody, and cells were

subsequently incubatedwith Alexa 488–labeled donkey anti-goatIgG (Invitrogen,Molecular Probes). Cell surface GPNMBproteinswere detected without membrane permeabilization. For IHC,bound antibodies were detected using ImmPRESS Reagent KitPeroxidase Anti-Goat IgG (Vector Laboratories).

Animal experimentsSix-week-old female NOD-SCID mice (CLEA) were subcuta-

neously injected with 5 � 106 Hs578T cells. Four weeks later, themice were sacrificed and fresh tumors were obtained for RNAisolation or IHC staining as described above.

For tumor formation using 4T1 cells, 1 � 106 4T1 cells wereinjected subcutaneously into 6-week-old female Balb/c mice(CLEA). After 3–4weeks, themice were sacrificed, and the tumorswere minced and dissociated under incubation conditions at37�C in 1 mg/mL collagenase (Wako) for 2 hours, 0.25% trypsin(Sigma) for 5 minutes, and 0.1 mg/mL DNase I (Roche) and5 mg/mL Dispase (Gibco) for 5 minutes. After centrifugation,single cells were obtained and the cell number was countedto perform FACS analysis and allograft transplantation for sec-ondary tumor formation. After 33 days, the mice were sacrificed,and the secondary tumors were collected. The tumor weight wasmeasured and the volumes were approximated using the follow-ing formula: volume¼ 0.5� a� b2, where a and b are the lengthsof the major and minor axes, respectively.

All the animal experiments were performed with the approvalof the Animal Ethics Committee of the University of Tsukuba(Ibaraki, Japan) and in accordance with the university's animalexperiment guidelines and the provisions of the Declaration ofHelsinki in 1995.

FACS analysisTwo-dimensional–cultured cells were treated with trypsin (Sig-

ma), and 3D-cultured spheres were dissociated using Accutasesolution (Gibco). Single cells were incubated with anti-GPNMBantibody (AF2550; R&D Systems), and then with Alexa 488–labeled donkey anti-goat IgG (Invitrogen, Molecular Probes) onice for 30minutes. The sampleswere analyzed using BD FACSAria(BD Biosciences) and BD FACSDiva software (100 mmSorp AriaII5B 3R 3V 5YG; BD Biosciences). We set the gating to collect cellsurface-GPNMBlow and -GPNMBhigh cells using the cells incubat-ed with Alexa 488–labeled donkey anti-goat IgG only as thenegative control.

Extreme limiting dilution analysis and sphere formation assayExtreme limiting dilution analysis (ELDA) was performed as

described previously (22). In brief, the breast cancer cells culturedin 2D or 3D culture conditions and the cell surface-GPNMBlow

and -GPNMBhigh subpopulations from 3D-cultured spheres wereseeded in a series of numbers from 200 cells/well to 1 cell/well in200-mL sphere culture medium and cultured for 14 days. Thenumber of wells containing spheres for each seeding cell numberwas counted and then analyzed using online ELDA software(http://bioinf.wehi.edu.au/software/elda).

Statistical analysisQuantitative data were expressed as mean � SEM. Statistical

analyses were performed using the t test or one-way ANOVA withthe Tukey multiple comparison test using GraphPad Prism 7software. P < 0.05 was considered significant.

GPNMB Induces Stemness in Dormant Breast Cancer Cells

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http://bioinf.wehi.edu.au/software/elda


ResultsGPNMB, CSC genes, EMT-TF genes, and mesenchymal markergenes are enriched in 3D cultures

We enriched breast cancer stem cells (BCSC) using the 3Dsphere culture method, which is used to examine the anchor-age-independent growth potential (23). Three different humanbreast cancer cell lines, BT-474 (luminal type), Hs578T (basaltype), and MDA-MB-468 (basal type), and a mouse breastcancer cell line, 4T1, were cultured in the 2D or 3D cultureconditions and compared for the expression levels of GPNMBmRNA together with those of known CSC genes such as SOX2,NANOG, OCT4, CD44, CD133, and FOXO3. All of these mRNAlevels were significantly higher in the 3D-cultured cells than in

the 2D-cultured cells (Fig. 1A–D; Supplementary Fig. S1A–S1D). We also examined CD24 mRNA expression becauseBCSCs are often characterized by CD44high/CD24low popula-tions in breast cancer cells (24, 25). The expression levels ofCD44mRNA were higher and those of CD24mRNA were lowerin the 3D-cultured cells than in the 2D-cultured cells (Fig. 1A–D, two panels from the right). In addition, we examined thesphere-forming frequencies of the 2D- and 3D-cultured Hs578Tcells by ELDA (22). ELDA provides an estimate of sphere-forming frequency using one-sided confidence intervals andstatistical analysis. The 3D cultures yielded significantly highersphere-forming frequency than did the 2D cultures (Supple-mentary Fig. S1E).

Figure 1.

Expression levels ofGPNMB and CSC genes in 2D or 3D culture conditions. A–D,mRNA expression levels ofGPNMB, SOX2, NANOG, OCT4, CD44, and CD24 in 2D- or3D-cultured BT-474 (A), Hs578T (B), MDA-MB-468 (C), and 4T1 (D) cells were analyzed by means of qPCR (mean � SEM; n ¼ 3). The results were normalized tob-actin levels. �� , P < 0.01; �� , P < 0.001; t test. Data are representative of three independent replicates.

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Furthermore, we evaluated the expression of EMT-TF genessuch as SNAIL, SLUG, and ZEB1 in BT-474, Hs578T, MDA-MB-468, and 4T1 cells. The expression levels of these EMT-TFmRNAs were also enhanced in the 3D-cultured cells as com-pared with the levels in the 2D-cultured cells (Fig. 2A–D).We also tested the expression of epithelial and mesenchymal

marker genes including CDH1, CDH2, fibronectin, and vimentinin Hs578T and 4T1 as well. An epithelial marker, Cdh1, wasdetected only in 4T1 cells and downregulated in the 3D cul-tures. On the other hand, mesenchymal marker genes wereenriched in the 3D-cultured both Hs578T and 4T1 cells (Sup-plementary Fig. S1F and S1G).

Figure 2.

Expression levels of EMT-TF genes in 2D or3D culture conditions. A–D, mRNAexpression levels ofSLUG, SNAIL, andZEB1 in2D- or 3D-cultured BT-474 (A), Hs578T (B),MDA-MD-468 (C), and 4T1 (D) cells wereanalyzed by means of qPCR (mean � SEM;n ¼ 3). The results were normalized tob-actin levels. � , P < 0.05; �� , P < 0.01;�� , P < 0.001; t test. Data are representativeof three independent replicates.






These results suggest that the 3D cultures can enrich the cellswith CSC-like properties in both human andmouse breast cancercell lines and that mRNA levels ofGPNMB correlate with those ofCSC genes, EMT-TF genes, andmesenchymalmarker genes in thisculture condition.

To examine the importance of GPNMB in the induction ofCSC genes and EMT-TF genes, we knocked down Gpnmb andexamined the expression levels of these genes in the 2D- and3D-cultured 4T1 cells. Knockdown of Gpnmb significantly sup-pressed the induction of CSC genes such as Sox2, Nanog, andCD44, and of EMT-TF genes such as Snail, Slug, and Zeb1, in the3D-cultured 4T1 cells (Supplementary Fig. S2A and S2B).Moreover, knockdown of Gpnmb reduced the sphere-formingfrequency of 4T1 cells (Supplementary Fig. S2C). Therefore,

GPNMB is critical for induction of CSC-like properties in the3D-cultured breast cancer cells.

Correlation between growth arrest and expressions of GPNMBand CSC genes and cell surface GPNMB protein exposure

We next evaluated the proliferative states of the cells indifferent culture conditions and in in vivo tumorigenic condi-tion because one of the characteristics of CSCs is their slowproliferation or dormancy. MKI67-positive and -negativeHs578T cell numbers were counted in the 2D and 3D culturesby using immunofluorescent staining and in xenograft tumorsby using IHC staining. The MKI67-positive and -negative ratiosin each condition are shown in Fig. 3A. More than 97% of the2D-cultured cells were MKI67-positive. However, nearly 80% of

Figure 3.

Correlation between proliferative states and expression levels of CSC genes and GPNMB. A, MKI67-positive and -negative cell numbers were counted afterimmunofluorescent staining of 2D- or 3D-cultured Hs578T cells. IHC staining was used for counting MKI67-positive and -negative cells in Hs578T xenograft tumors.mRNAexpression levels ofMKI67 (B), SOX2, andGPNMB (C) in 2D- or 3D-culturedHs578T andHs578T tumorswere analyzedbymeans of qPCR (mean�SEM; n¼ 3).The results were normalized to b-actin levels. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001; ANOVA with the Tukey multiple comparison test; n.s.,not significant. Data are representative of three independent replicates. D, Hs578T cells were cultured in subconfluent or confluent 2D culture conditions.Before being harvested, cells were serum-starved for 48 hours. mRNA expression levels of SOX2, NANOG, and GPNMB were analyzed by means of qPCR(mean� SEM; n¼ 3). The results were normalized to b-actin levels. �� , P < 0.01; t test; n.s., not significant. Data are representative of three independent replicates.E, Immunoblot analysis was performed to detect GPNMB protein in 2D- or 3D-cultured Hs578T cells. b-Actin was used as the loading control. Data arerepresentative of more than three independent replicates. F and G, Cell surface GPNMB was detected by FACS. Percentages of cell surface-GPNMBlow and-GPNMBhigh subpopulations of Hs578T cells in 2D (F) or 3D (G) cultures. Data are representative of more than three independent replicates.

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Figure 4.

GPNMB is exposed on cell surface of dormant breast cancer stem-like cells. A, Schema of experimental procedures. Breast cancer cells were cultured in3D culture condition and cell surface-GPNMBlow and -GPNMBhigh cells were sorted by FACS. Each population was used for ELDA or RNA isolation. B, Sphere-forming frequencies of cell surface-GPNMBlow and -GPNMBhigh cells in 3D-cultured Hs578T spheres were examined by ELDA. n ¼ 8 for each point. Data arerepresentative of three independent replicates. C–E,mRNA expression levels ofGPNMB, SOX2, NANOG (C) SNAIL, SLUG (D), andMKI67 and TPX2 (E) in 2D-culturedHs578T cells and in cell surface-GPNMBlow and -GPNMBhigh cells of 3D-cultured Hs578T spheres were analyzed by means of qPCR (mean � SEM; n ¼ 3). Theresults were normalized to b-actin levels. � , P <0.05; �� , P < 0.01; �� , P < 0.001; �� , P <0.0001; ANOVAwith the Tukeymultiple comparison test; n.s., not significant;n.d., not detected. Data are representative of three independent replicates.






the 3D-cultured spheres and nearly 50% of the xenografttumors were MKI67-negative. In addition, abundance ofMKI67mRNA was significantly higher in the 2D-cultured cells than inthe 3D-cultured spheres or xenograft tumors (Fig. 3B). Inter-estingly, SOX2 and GPNMB mRNA expression was enriched inthe spheres and tumors, and highly correlated with the MKI67-negative cell ratios, suggesting that dormant cells may havehigher expression levels of SOX2 and GPNMB (Fig. 3C). Toconfirm whether growth arrest affects the expression of CSCgenes and GPNMB, we induced growth arrest by serum star-vation for 48 hours under subconfluent or confluent 2D cultureconditions. Quantitative data obtained by qPCR showed thatthe mRNA expression levels of CSC genes SOX2 and NANOG aswell as GPNMB were increased in serum-free growth-arrestedconditions, especially under confluent cell density (Fig. 3D).On the other hand, GPNMB protein was relatively abundanteven in the 2D-cultured Hs578T cells without starvation(Fig. 3E; Supplementary Fig. S3A) when compared with thesignificantly higher induction of GPNMB mRNA in the 3Dcultures than in the 2D cultures (Fig. 1B, left). However, flowcytometry and immunofluorescent staining with/without per-meabilization revealed that less than 1% of the 2D-culturedHs578T cells had only low levels of cell surface GPNMB (Fig.3F), whereas nearly 10% of the cells were cell surface GPNMB-positive in the 3D-cultured Hs578T cells (Fig. 3G; Supplemen-tary Fig. S3B). Similar regulation of GPNMB mRNA expressionand cell surface protein exposure were confirmed with otherbreast cancer cell lines, BT-474 and MDA-MB-468. These results

suggest that growth-arrested conditions such as those afterserum starvation for confluent 2D cultures or 3D cultures canactivate the sorting of GPNMB protein for its exposure on thecell surface together with enhanced GPNMB transcription andCSC genes' induction.

GPNMB is exposed on the surface of dormant breast cancerstem-like cells in 3D-cultured spheres

To characterize the cell surface GPNMB-positive breast cancercells, we isolated cell surface-GPNMBlow and -GPNMBhigh cellpopulations from the 3D-cultured Hs578T spheres by FACS,and the sphere-forming frequencies were compared in thesetwo populations by use of ELDA together with cell proliferationand CSC genes expression by qPCR (Fig. 4A). ELDA determinedthat approximately 1 in 76 cell surface-GPNMBhigh cells har-bored sphere-forming stem cell potential, while the cell surface-GPNMBlow cells had much less frequency (about 1 in 1,955;Fig. 4B). These results indicate significant differences in sphere-forming frequencies between the populations of the cell sur-face-GPNMBlow and -GPNMBhigh cells. We further comparedthe mRNA abundance of GPNMB and CSC genes SOX2 andNANOG in the 2D-cultured cells and in the cell surface-GPNMBlow and -GPNMBhigh cells of 3D-cultured spheres. Boththe cell surface-GPNMBlow and cell surface-GPNMBhigh cellshad higher expression levels of GPNMB, SOX2, and NANOGthan did the 2D-cultured cells. Obviously, the cell surface-GPNMBhigh population had much higher SOX2 and NANOGthan did the cell surface-GPNMBlow population. However, the

Figure 5.

Cell surface-GPNMBhigh dormant breast cancer cells possess stem cell–like properties. A, ELDA was performed to determine sphere-forming frequencies of cellsurface-GPNMBlow and -GPNMBhigh cells in 3D-cultured 4T1 spheres. n ¼ 8 for each point. Data are representative of two independent replicates. B–D, mRNAexpression levels of Gpnmb, Sox2, Nanog (B), Snail, Slug (C), and Mki67 (D) in 2D-cultured 4T1 cells and in cell surface-GPNMBlow and -GPNMBhigh cells of3D-cultured 4T1 spheres were analyzed by means of qPCR (mean � SEM; n ¼ 3). The results were normalized to b-actin levels. �� , P < 0.01; �� , P < 0.001;�� , P < 0.0001; ANOVA with the Tukey multiple comparison test; n.s., not significant. Data are representative of three independent replicates.

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GPNMB mRNA expression levels did not differ significantlybetween the cell surface-GPNMBlow and -GPNMBhigh cells(Fig. 4C). Moreover, the expression levels of EMT-TF genesSNAIL and SLUG, EMT markers CDH2, fibronectin, and vimentin,and the proliferation marker genes MKI67 and TPX2 (26)were examined subsequently. The expression levels of SNAILand SLUG (Fig. 4D) as well as of CDH2, fibronectin, and vimentin(Supplementary Fig. S4A) were highly correlated with thoseof SOX2 and NANOG. In contrast, the 2D-cultured cells hadthe highest expression levels of proliferation marker genes,while the cell surface-GPNMBhigh cells showed no detectablelevels of MKI67 or TPX2 (Fig. 4E).

In addition, we confirmed a similar phenomenon in amouse breast cancer cell line 4T1. ELDA results showed thatthe cell surface-GPNMBhigh cells had higher sphere-forming

potential, while the cell surface-GPNMBlow cells had less fre-quency (Fig. 5A). Next, we compared the mRNA abundance ofGpnmb, Sox2, and Nanog in the 2D-cultured cells and in the cellsurface-GPNMBlow and -GPNMBhigh cells of 3D-culturedspheres. The expression levels of Gpnmb, Sox2, and Nanog inthe cell surface-GPNMBhigh cells were significantly higher thanthose in the 2D-cultured cells or the cell surface-GPNMBlow

cells (Fig. 5B). Moreover, the cell surface-GPNMBhigh cellsshowed enhanced expression levels of Snail, Slug (Fig. 5C),fibronectin, and vimentin (Supplementary Fig. S4B, middleand right), whereas they showed the lowest levels of Mki67(Fig. 5D) and Cdh1 (Supplementary Fig. S4B, left). Takentogether, these results suggest that the cell surface-GPNMBhigh

cells have the properties of dormant CSCs in the 3D-cluturedboth human and mouse breast cancer cell lines.

Figure 6.

GPNMB is exposed on cell surface of dormant cancer stem–like cells derived frombreast tumors.A,ELDAwasperformed to determine sphere-forming frequencies ofcell surface-GPNMBlow and -GPNMBhigh cells in 4T1 allograft tumors. n ¼ 8 for each point. Data are representative of two independent replicates. mRNAexpression levels ofGpnmb, Sox2, Nanog (B), Snail, Slug (C), andMki67 (D) in bulk tumor cells (indicated as "Tumor") and in cell surface-GPNMBlow and -GPNMBhigh

cells of 4T1 allograft tumorswere analyzed bymeans of qPCR (mean�SEM; n¼ 3). The resultswere normalized tob-actin levels. � ,P<0.05; �� ,P <0.01; �� ,P <0.001;�� , P < 0.0001; ANOVA with the Tukey multiple comparison test; n.s., not significant. Data are representative of three independent replicates. E and F, Secondarytumor-forming frequencies were examined. Tumor cells derived from 4T1 allograft tumors were sorted by FACS into cell surface-GPNMBhigh and -GPNMBlow

subpopulations and then injected into mice. Secondary tumors excised on 33 days after transplantation (E). Secondary tumor incidences of the cell surface-GPNMBhigh and -GPNMBlow 4T1 tumor cells (F). n¼ 5.G andH,Cell surfaceGPNMBwas detected by FACS. Percentages of the cell surface-GPNMBlow and -GPNMBhigh

subpopulations in 4T1 tumor cells (G) or secondary tumors derived from the cell surface-GPNMBlow or -GPNMBhigh 4T1 tumor cells (H).






GPNMB is exposed on the surface of dormant breast cancerstem-like cells in allograft tumors and enhances tumorigenicity

We further examined the characteristics of the cell surface-GPNMBlow and -GPNMBhigh cells using a 4T1 allograft tumormodel. We compared the mRNA abundance of Gpnmb, Sox2, andMki67 in the 2Dor 3D-cultured 4T1 cells and 4T1 tumors. The 4T1tumors had lower expression levels of Gpnmb and Sox2 than didthe 3D-cultured spheres, but those levels were still more enrichedthan those of the 2D-cultured cells (Supplementary Fig. S5A).Mki67 expression levels were lower in the 3D-cultured cells ortumors than in the 2D-cultured cells, similar to Hs578T tumors(Supplementary Fig. S5B). After harvesting 4T1 tumors, we col-

lected isolated single cells from the tumors and sorted cell surface-GPNMBlow and -GPNMBhigh cells by FACS. ELDA showed that thecell surface-GPNMBhigh cells harbored higher sphere-formingfrequency (about 1 stem cell in 29 cells), while the cell surface-GPNMBlow cells had much less sphere-forming frequency (about1 in 1,065; Fig. 6A). Moreover, the cell surface-GPNMBhigh tumorcells showed the highest expression levels of Gpnmb, Sox2, Nanog(Fig. 6B), Snail, and Slug (Fig. 6C), and the lowest expression levelof Mki67 mRNA (Fig. 6D). These results indicated that like3D-cultured spheres, the tumors grown in vivo are also composedof cell surface-GPNMBlow and -GPNMBhigh cells and that the cellsurface-GPNMBhigh cells have dormant CSC-like properties.

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Figure 7.

The tyrosine residue in hemITAM of GPNMB is essential for induction of CSC genes. A, mRNA expression levels of Gpnmb, Sox2, and Nanog in NMuMG-mock,NMuMG-GPNMB, and NMuMG-GPNMB (YF) cells cultured in 2D or 3D culture conditions were analyzed by means of qPCR (mean � SEM; n ¼ 3). The resultswere normalized to b-actin levels. � , P < 0.05; �� , P < 0.001; �� , P < 0.0001; ANOVA with the Tukey multiple comparison test; n.s., not significant; n.d.,not detected. Data are representative of three independent replicates. B, Graphic illustration of the functional roles of GPNMB in breast cancer cells. Cellsurface GPNMB induces the expression of CSC genes and EMT-TF genes in dormant breast cancer cells and contributes to the tumorigenicity.

Chen et al.





Furthermore, we tested the secondary tumor growth of the cellsurface-GPNMBlow and -GPNMBhigh 4T1 tumor cells. Injected cellsurface-GPNMBhigh cells generated secondary tumors with ahigher incidence rate (103, 3/5; 104, 5/5) than that of the cellsurface-GPNMBlow cells (103, 1/5; 104, 1/5; Fig. 6E and F;Supplementary Fig. S5C and S5D). These results clearly indicatedthat the cell surface-GPNMBhigh cells have higher secondarytumor-forming frequency. In addition, further analysis of thetumors generated by the cell surface-GPNMBlow cells indicatedthat these tumorswere also composedof a comparable ratioof cellsurface-GPNMBhigh and -GPNMBlow cells, with the tumor madefrom the cell surface-GPNMBhigh cells, suggesting that the plas-ticity of cancer cells generate cell surface-GPNMBhigh cells fromthe cell surface-GPNMBlow cells at low frequency and causes thetumorigenic potential (Fig. 6G and H).

The tyrosine residue in GPNMB hemITAM is essential forinduction of CSC genes

To elucidate the molecular mechanism of GPNMB in genera-tion of CSC-like properties, we examined the involvement of thetyrosine residue in hemITAM, because we have previously dem-onstrated that Tyr529 is essential for GPNMB-inducible EMT andtumorigenesis. We used NMuMG-mock, NMuMG-GPNMB, andNMuMG-GPNMB(YF) cell lines, in which Tyr529 is replaced by aphenylalanine (14). Exogenous Gpnmb mRNA was highlyexpressed in both NMuMG-GPNMB and NMuMG-GPNMB(YF)cells cultured in the 2D and 3D culture conditions (Fig. 7A, left;Supplementary Table S2, top), but the expression of the CSCgenes Sox2 and Nanog was induced in NMuMG-GPNMB cellscultured in the 3D culture condition only. On the other hand,GPNMB(YF) cells failed to induce the expression of Sox2 andNanog mRNA, even in the 3D culture condition (Fig. 7A, middleand right; Supplementary Table S2, middle and bottom). Theseresults indicate that GPNMB function mediated by the tyrosineresidue in hemITAM is essential for the induction of CSC genes inthe 3D cultures.

DiscussionIn this article, we have reported a novel function of GPNMB,

which is exposed on the surface of growth-arrested breast cancercells and induces stem cell–like properties through hemITAM(Fig. 7B).

We previously demonstrated that GPNMB can induce EMT inNMuMG cells (14). The correlation between EMT and the acqui-sition of stem cell-like properties has been proven in breastepithelial cells (15), and EMT-TFs, including TWIST, SLUG,SNAIL, ZEB1, and ZEB2, have been shown to confer the char-acteristics of CSCs (15, 27, 28). In addition, Chen and colleaguesshowed that 3D-cultured cells had higher expression of the EMT-TF genes SNAIL and TWIST than did 2D-cultured cells in head andneck squamous cell carcinoma cell lines (29). The 3D culturesystem is thought to mimic the cellular dynamics in the bodybetter than the 2D culture system (30–32) and is used to enrichcellswith stemcell potential in cancers of thebreast (33, 34), brain(35), and head and neck (29). In our 3D cultures, the spheres hadenhanced expression levels of CSC genes as well as of EMT-TFgenes and altered those of EMT marker genes (Figs. 1, 2, 4C andD, 5B andC, and 7A; Supplementary Fig. S1A–S1D, S1F, S1G, andS4). Importantly, similar results were observed in the xenograftand allograft tumors (Figs. 3C, 6B and C; Supplementary

Fig. S5A). Taken together, these results indicate that the 3D culturemethod is a useful in vitro system to examine CSC-like properties.

CSCs are found in acute myeloid leukemia (36) and solidtumors including those of the breast (24), brain (37), prostate(38), colon (39), pancreas (40), and lung (41). Because BCSCswere isolated as cells with themarkers of CD44þ/CD24�/low/Lin�

(24), CD44þ/CD24� is frequently used as a BCSC marker (25).Other surface markers, such as CD133, CD49f, and CD61, havealso been reported (42–45).Whereas GPNMBwas locatedmainlyon lysosome or endosomemembranes in 2D-cultured cells, it wasexposed on the cell surface of dormant BCSCs and induced stemcell-like properties in 3D-cultured spheres and in vivo tumors(Figs. 3F and G, 4B–E, 5, and 6). Our results indicate that surfaceexpression of GPNMB could be a novel indicator of dormantBCSCs. However, the frequency with which stem cells wereenriched with cell surface-GPNMBhigh cells in the 3D-culturedspheres or tumorswas not 100% (Figs. 4B, 5A, and 6A). Therefore,investigation of possible and more suitable combinations ofBCSC markers might be required for further characterization ofbreast cancer stem-like cells.

Although we have not elucidated the mechanism wherebyGPNMB is exposed on the surface of dormant cells, we havehere shown that growth-arrested conditions, such as serumstarvation or 3D culture conditions, induced transcriptionalactivation of GPNMB and total GPNMB protein (Fig. 3D–G;Supplementary Fig. S3). In addition, GPNMB is exposed on thecell surface only in MKI67-negative growth-arrested cells(Figs. 4E, 5D, and 6D). Moreover, cell growth analyses shownin Fig. 3A indicated that the 2D cultures mostly composed ofmonotonous MKI67-positive proliferating cells. On the otherhand, the 3D cultures and in vivo tumors contain both growth-arrested cells and proliferating cells. This divergence in cellproliferation status is thought to make the critical difference in2D-cultured cells and 3D-cultured spheres or in vivo tumors.Qian and colleagues have reported that inhibition of the ERKsignaling pathway enhances GPNMB protein expression inseveral melanoma cell lines that have NRAS or BRAF mutations(46). Taken together, these findings suggest that growth inhi-bition may enhance GPNMB transcriptional activation andprotein sorting to the cell surface, and leads to maintenanceof CSC-like phenotypes.

GPNMB contains hemITAM (YxxI) and a dileucine motif (D/ExxLL) in its cytoplasmic tail. Thesemotifs are frequently found intransmembrane proteins and function as sorting signals associ-ated with endocytosis or endosomal/lysosomal membrane traf-ficking (47). Our previous study demonstrated that hemITAM isessential for inducing EMT and tumorigenesis via phosphoryla-tion of the tyrosine residue by SRC kinase (14). We have herefurther shown that it is involved in the induction of CSC genesin the 3D cultures (Fig. 7A). The loss tumorigenicity madeit difficult to analyze the gene expression levels of mutanthemITAM-expressing tumors in vivo. Moreover, Lin and collea-gues reported that cell surface GPNMB forms a heterodimer withEGFR and that the tyrosine residue in hemITAM is phosphory-lated upon heparin-binding EGF (HB-EGF) stimulation. Subse-quently, the BRK– or LRRK2–LINK-A complex binds to GPNMBand stabilizes normoxic HIF1a. They also showed that phosphor-ylation of GPNMB is detected in TNBC and correlates with themetastatic status of patients with breast cancer (48). We have alsoshown that EGFR and FGFR1 were enriched in the 3D-culturedHs578T and 4T1 cells (Supplementary Fig. S6A and S6B).






Therefore, it is worthwhile examining the cross-talk betweentyrosine receptor kinase signaling and GPNMB in detail. Theseresults together with our current observations suggest that bothcell surface expression of GPNMB and phosphorylation of thetyrosine residue in hemITAM are essential for the GPNMB-induc-ible tumorigenic andmetastatic potential, at least partly owing tothe generation of CSC-like properties.

GPNMB is known to have a soluble form shed by ADAM10(49), and it could be detected in patients' blood (8). Therefore,shedding of GPNMB is thought to happen on the cell surface.So far we know of no relationship among GPNMB cell surfacelocalization, hemITAM tyrosine phosphorylation, and shed-ding of its extracellular domain and it would be interesting tostudy these points as a serial phenomenon to understand thephysiologic and pathologic functions of GPNMB.

CDX-011 (glembatumumab vedotin) is an antibody–drugconjugate that contains CR011, a human mAb against GPNMB,and conjugates with a cytotoxic agent (MMAE). CDX-011 hasbeen developed for the treatment of GPNMB-expressing cancers,and clinical studies ofCDX-011 against patientswithbreast cancerandmelanoma were conducted (12, 13, 50). We have shown thatthe cell surface-GPNMBhigh cells have higher stem cell–like prop-erties than do the cell surface-GPNMBlow cells (Fig. 7B). Thus, ournovel findings lead us to propose that targeting GPNMB, which isexposed on the surface of dormant cancer cells, has better feasi-bility to kill CSCs and might result in a highly efficient cancertreatment.

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

Authors' ContributionsConception and design: C. Chen, Y. Okita, M. KatoDevelopment of methodology: C. Chen, Y. Okita, F. Abe, M. KatoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Chen, Y. Okita, Y. Watanabe, F. Abe, M.A. Fikry,Y. Ichikawa, A. Shibuya, M. KatoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Chen, Y. Okita, F. Abe, M. KatoWriting, review, and/or revision of the manuscript: C. Chen, Y. Okita,H. Suzuki, M. KatoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. SuzukiStudy supervision: Y. Okita, M. Kato

AcknowledgmentsWe thank F. Miyamasu (Medical English Communication Center, University

of Tsukuba, Ibaraki, Japan) for proofreading the manuscript. This research wassupported by JSPSGrantNumbers JP15K19070, JP17K14981, and JP18H02676and grants from the Naito Foundation and the Uehara Memorial Foundation.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 23, 2018; revised July 31, 2018; accepted September 12,2018; published first September 17, 2018.

References1. Dai X, Li T, Bai Z, Yang Y, Liu X, Zhan J, et al. Breast cancer intrinsic

subtype classification, clinical use and future trends. Am J Cancer Res2015;5:2929–43.

2. De Laurentiis M, Cianniello D, Caputo R, Stanzione B, Arpino G, Cinieri S,et al. Treatment of triple negative breast cancer (TNBC): current options andfuture perspectives. Cancer Treat Rev 2010;36,Suppl 3:S80–6.

3. Jia LY, Shanmugam MK, Sethi G, Bishayee A. Potential role of targetedtherapies in the treatment of triple-negative breast cancer. AnticancerDrugs2016;27:147–55.

4. Kuan CT, Wakiya K, Dowell JM, Herndon JE II, Reardon DA, Graner MW,et al. Glycoprotein nonmetastatic melanoma protein B, a potential molec-ular therapeutic target in patients with glioblastoma multiforme.Clin Cancer Res 2006;12:1970–82.

5. TseKF, JeffersM, Pollack VA,McCabeDA, ShadishML,KhramtsovNV, et al.CR011, a fully humanmonoclonal antibody-auristatin E conjugate, for thetreatment of melanoma. Clin Cancer Res 2006;12:1373–82.

6. Rose AA, Pepin F, Russo C, Abou Khalil JE, Hallett M, Siegel PM. Osteoac-tivin promotes breast cancer metastasis to bone. Mol Cancer Res2007;5:1001–14.

7. Rose AA, Grosset AA, Dong Z, Russo C, Macdonald PA, Bertos NR, et al.Glycoprotein nonmetastatic B is an independent prognostic indicator ofrecurrence and a novel therapeutic target in breast cancer. Clin Cancer Res2010;16:2147–56.

8. Kanematsu M, Futamura M, Takata M, Gaowa S, Yamada A, Mor-imitsu K, et al. Clinical significance of glycoprotein nonmetastatic Band its association with HER2 in breast cancer. Cancer Med 2015;4:1344–55.

9. Zhou LT, Liu FY, Li Y, Peng YM, Liu YH, Li J. Gpnmb/osteoactivin, anattractive target in cancer immunotherapy. Neoplasma 2012;59:1–5.

10. Maric G, Rose AA, Annis MG, Siegel PM. Glycoprotein non-metastatic b(GPNMB): a metastatic mediator and emerging therapeutic target incancer. Onco Targets Ther 2013;6:839–52.

11. Vaklavas C, Forero A.Management ofmetastatic breast cancer with second-generation antibody-drug conjugates: focus on glembatumumab vedotin(CDX-011, CR011-vcMMAE). BioDrugs 2014;28:253–63.

12. Rose AA, Biondini M, Curiel R, Siegel PM. Targeting GPNMB with glem-batumumab vedotin: current developments and future opportunities forthe treatment of cancer. Pharmacol Ther 2017;179:127–41.

13. Taya M, Hammes SR. Glycoprotein non-metastatic melanoma protein B(GPNMB) and cancer: a novel potential therapeutic target. Steroids2018;133:102–7.

14. Okita Y, Kimura M, Xie R, Chen C, Shen LT, Kojima Y, et al. Thetranscription factor MAFK induces EMT and malignant progression oftriple-negative breast cancer cells through its target GPNMB. Sci Signal2017;10:eaak9397.

15. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al. Theepithelial-mesenchymal transition generates cells with properties of stemcells. Cell 2008;133:704–15.

16. Scheel C, Weinberg RA. Cancer stem cells and epithelial-mesenchymaltransition: concepts and molecular links. Semin Cancer Biol 2012;22:396–403.

17. Sato R, Semba T, SayaH, ArimaY.Concise review: Stem cells and epithelial-mesenchymal transition in cancer: Biological implications and therapeutictargets. Stem Cells 2016;34:1997–2007.

18. Al-Hajj M, Clarke MF. Self-renewal and solid tumor stem cells. Oncogene2004;23:7274–2.

19. MimeaultM, Batra SK.Newadvances on critical implicationsof tumor- andmetastasis-initiating cells in cancer progression, treatment resistance anddisease recurrence. Histol Histopathol 2010;25:1057–73.

20. Bozorgi A, Khazaei M, Khazaei MR. New findings on breast cancer stemcells: a review. J Breast Cancer 2015;18:303–12.

21. Okita Y, Kamoshida A, Suzuki H, Itoh K, Motohashi H, Igarashi K, et al.Transforming growth factor-b induces transcription factors MafK andBach1 to suppress expression of the heme oxygenase-1 gene. J Biol Chem2013;288:20658–67.

22. Hu Y, Smyth GK. ELDA: extreme limiting dilution analysis for comparingdepleted and enriched populations in stem cell and other assays. J ImmunolMethods 2009;347:70–8.

23. Weiswald LB, Bellet D, Dangles-Marie V. Spherical cancer models in tumorbiology. Neoplasia 2015;17:1–15.

Chen et al.





24. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF.Prospective identification of tumorigenic breast cancer cells. Proc NatlAcad Sci U S A 2003;100:983–8.

25. Kai K, Arima Y, Kamiya T, Saya H. Breast cancer stem cells. Breast Cancer2010;17:80–5.

26. Brizova H, Kalinova M, Krskova L, Mrhalova M, Kodet R. A novel quan-titative PCR of proliferation markers (Ki-67, topoisomerase IIalpha, andTPX2): an immunohistochemical correlation, testing, and optimizing formantle cell lymphoma. Virchows Arch 2010;456:671–9.

27. Guo W, Keckesova Z, Donaher JL, Shibue T, Tischler V, Reinhardt F, et al.Slug and Sox9 cooperatively determine the mammary stem cell state. Cell2012;148:1015–28.

28. Chu PY, Hu FW, Yu CC, Tsai LL, Yu CH, Wu BC, et al. Epithelial-mesenchymal transition transcription factor ZEB1/ZEB2 co-expressionpredicts poor prognosis andmaintains tumor-initiating properties in headand neck cancer. Oral Oncol 2013;49:34–41.

29. Chen C, Wei Y, Hummel M, Hoffmann TK, Gross M, Kaufmann AM, et al.Evidence for epithelial-mesenchymal transition in cancer stem cells of headand neck squamous cell carcinoma. PLoS One 2011;6:e16466.

30. Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ,et al. In vitropropagation and transcriptional profiling of humanmammarystem/progenitor cells. Genes Dev 2003;17:1253–70.

31. Yamada KM, Cukierman E. Modeling tissue morphogenesis and cancer in3D. Cell 2007;130:601–10.

32. Fatehullah A, Tan SH, Barker N. Organoids as an in vitro model of humandevelopment and disease. Nat Cell Biol 2016;18:246–54.

33. Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, et al.Isolation and in vitro propagation of tumorigenic breast cancer cells withstem/progenitor cell properties. Cancer Res 2005;65:5506–11.

34. Yu F, Yao H, Zhu P, Zhang X, Pan Q, Gong C, et al. let-7 regulates self-renewal and tumorigenicity of breast cancer cells. Cell 2007;131:1109–23.

35. Ghods AJ, Irvin D, Liu G, Yuan X, Abdulkadir IR, Tunici P, et al. Spheresisolated from 9L gliosarcoma rat cell line possess chemoresistant andaggressive cancer stem-like cells. Stem Cells 2007;25:1645–53.

36. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as ahierarchy that originates from a primitive hematopoietic cell. Nat Med1997;3:730–7.

37. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, et al.Identification of a cancer stem cell in human brain tumors. Cancer Res2003;63:5821–8.

38. Patrawala L, Calhoun T, Schneider-Broussard R, LiH, Bhatia B, Tang S, et al.Highly purifiedCD44þprostate cancer cells fromxenograft human tumors

are enriched in tumorigenic and metastatic progenitor cells. Oncogene2006;25:1696–708.

39. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C,et al. Identification and expansion of human colon-cancer-initiating cells.Nature 2007;445:111–5.

40. Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, et al. M.Identification of pancreatic cancer stem cells. Cancer Res 2007;67:1030–7.

41. Eramo A, Lotti F, Sette G, Pilozzi E, Biffoni M, Di Virgilio A, et al.Identification and expansion of the tumorigenic lung cancer stem cellpopulation. Cell Death Differ 2008;15:504–14.

42. Wright MH, Calcagno AM, Salcido CD, Carlson MD, Ambudkar SV,Varticovski L. Brca1 breast tumors contain distinct CD44þ/CD24� andCD133þ cells with cancer stem cell characteristics. Breast Cancer Res2008;10:R10.

43. Cariati M, Naderi A, Brown JP, Smalley MJ, Pinder SE., Caldas C, et al.Alpha-6 integrin is necessary for the tumourigenicity of a stem cell-likesubpopulation within the MCF7 breast cancer cell line. Int J Cancer2008;122:298–304.

44. Meyer MJ, Fleming JM, Lin AF, Hussnain SA, Ginsburg E, Vonderhaar BK.CD44posCD49fhiCD133/2hi defines xenograft-initiating cells in estrogenreceptor-negative breast cancer. Cancer Res 2010;70:4624–33.

45. Vaillant F, Asselin-Labat ML, Shackleton M, Forrest NC, Lindeman GJ,Visvader JE. The mammary progenitor marker CD61/beta3 integrin iden-tifies cancer stem cells in mouse models of mammary tumorigenesis.Cancer Res 2008;68:7711–7.

46. Qian X, Mills E, Torgov M, LaRochelle WJ, Jeffers M. Pharmacologicallyenhanced expression of GPNMB increases the sensitivity of melanomacells to the CR011-vcMMAE antibody-drug conjugate. Mol Oncol 2008;2:81–93.

47. Pandey KN. Functional roles of short sequencemotifs in the endocytosis ofmembrane receptors. Front Biosci 2009;14:5339–60.

48. Lin A, Li C, Xing Z,HuQ, Liang K,Han L, et al. The LINK-A lncRNA activatesnormoxic HIF1alpha signalling in triple-negative breast cancer. Nat CellBiol 2016;18:213–24.

49. Rose AAN, Annis MG, Dong Z, Pepin F, Hallett M, Park M, et al. ADMA10releases a soluble form of the GPNMB/Osteoactivin extracellular domainwith angiogenic properties. PLoS One 2010;5:e12093.

50. Yardley DA, Weaver R, Melisko ME, Saleh MN, Arena FP, Forero A, et al.EMERGE: A randomized phase II study of the antibody-drug conjugateglembatumumab vedotin in advanced glycoprotein NMB-expressingbreast cancer. J Clin Oncol 2015;33:1609–19.






2018;78:6424-6435. Published OnlineFirst September 17, 2018.Cancer Res Chen Chen, Yukari Okita, Yukihide Watanabe, et al.

like Properties−Cancer Cells and Induces Stem Cell Glycoprotein nmb Is Exposed on the Surface of Dormant Breast

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