Glycoprotein Nmb Is Exposed on the Surface of Dormant Breast Cancer Cells and Induces Stem Cell–Like Properties

Published OnlineFirst September 17, 2018; DOI: 10.1158/0008-5472.CAN-18-0599

Cancer Tumor Biology and Immunology Research

Glycoprotein nmb Is Exposed on the Surface of Dormant Breast Cancer Cells and Induces Stem Cell–like Properties Chen 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 transmembrane GPNMBhigh and -GPNMBlow cells from the spheres. Cell sur- protein that contributes to the initiation and malignant pro- face-GPNMBhigh cells expressed high levels of CSC genes and gression of breast cancer through induction of epithelial– EMT-TF genes, had significantly higher sphere-forming fre- mesenchymal transition (EMT). Although it is known that quencies than the cell surface-GPNMBlow cells, and showed no EMT is associated with not only cancer invasion but also detectable levels of proliferation marker genes. Similar results acquisition of cancer stem cell (CSC) properties, the function were obtained from transplanted breast tumors. Furthermore, of GPNMB in this acquisition of CSC properties has yet wild-type GPNMB, but not mutant GPNMB (YF), which lacks to be elucidated. To address this issue, we utilized a three- tumorigenic activity, induced CSC-like properties in breast dimensional (3D) sphere culture method to examine the epithelial cells. These findings suggest that GPNMB is exposed correlation between GPNMB and CSC properties in breast on the surface of dormant breast cancer cells and its activity cancer cells. Three-dimensional sphere cultures induced higher contributes to the acquisition of stem cell–like properties. expression of CSC genes and EMT-inducing transcription factor (EMT-TF) genes than the 2D monolayer cultures. Significance: These findings suggest that cell surface expres- Three-dimensional culture also induced cell surface expression sion of GPNMB could serve as a marker and promising of GPNMB on limited numbers of cells in the spheres, whereas therapeutic target of breast cancer cells with stem cell-like the 2D cultures did not. Therefore, we isolated cell surface- properties. Cancer Res; 78(22); 6424–35. 2018 AACR.

Introduction pies for patients with TNBC, further molecular characterization of TNBC is required (2, 3). Breast cancer is the most common cancer among women Glycoprotein nmb (GPNMB) is a type I transmembrane pro- worldwide, which is a heterogeneous disease and can be classified tein. Abundant expression of GPNMB was observed in glioblas- into five molecular subtypes, luminal A, luminal B, HER2- toma, melanoma, and breast cancer, especially in TNBC, and was enriched, basal-like, and normal-like, on the basis of the expres- reported as a prognostic factor (4–7). Moreover, the association sion of estrogen receptors (ER), progesterone receptors (PR), and between GPNMB- and HER2-positive breast cancers has also been HER2 (1). The basal-like subtype is also referred to as triple- reported (8). GPNMB is known to have functions in angiogenesis, negative breast cancer (TNBC) because it is typically negative for tumorigenesis, cell migration, and cell invasion and metastasis, ER, PR, and HER2. TNBC accounts for 10%–30% of all diagnosed and has received much attention as a target molecule for cancer breast cancers. In general, TNBC is associated with poor prognosis treatment (9–13). and high lethality. Moreover, the lack of effective molecularly We previously demonstrated that enhanced expression of targeted drugs limits the treatment options for this aggressive GPNMB induces epithelial–mesenchymal transition (EMT) and disease. Therefore, to develop novel molecularly targeted thera- increases anchorage-independent sphere formation and invasive tumor growth in vivo through the hemi-immunoreceptor tyro- – 1Department of Experimental Pathology, Graduate School of Comprehensive sine based activation motif (hemITAM) in the intracellular Human Sciences and Faculty of Medicine, University of Tsukuba, Ibaraki, Japan. domain. Furthermore, knockdown of GPNMB attenuated the 2Department of General Surgery, Xin Hua Hospital affiliated to Shanghai Jiao tumorigenic abilities of TNBC cell lines (14). EMT is an essential Tong University School of Medicine, Shanghai, China. 3Division of Cell Dynamics, process during embryogenesis, tissue repair, fibrosis, and cancer Transborder Medical Research Center, University of Tsukuba, Ibaraki, Japan. 4 invasion and metastasis. In 2008, Mani and colleagues reported Laboratory of Immunology, Life Science Center for Survival Dynamics of that the EMT-inducing transcription factors (EMT-TF) SNAIL and Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki, Japan. TWIST as well as TGFb signaling are associated with acquisition of Note: Supplementary data for this article are available at Cancer Research cancer stem cell (CSC) properties in breast epithelial cells (15). Online (http://cancerres.aacrjournals.org/). Evidence of CSC properties induced by EMT-TFs was also shown Corresponding Author: Yukari Okita, University of Tsukuba, 1-1-1 Tennodai, in breast cancer (16, 17). CSCs harbor the potential of self- Tsukuba, Ibaraki 305-8575, Japan. Phone/Fax: 81-29-853-3944; E-mail: renewal, differentiation, tumorigenesis, and resistance to drugs [email protected] or radiation owing to their ability to enter dormancy and their doi: 10.1158/0008-5472.CAN-18-0599 abundant expression of drug exporters (18–20). For these reasons, 2018 American Association for Cancer Research. CSCs are thought to be the root cause of cancer metastasis and

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GPNMB Induces Stemness in Dormant Breast Cancer Cells

relapse. Therefore, targeting EMT-related molecules might be a subsequently incubated with Alexa 488–labeled donkey anti-goat promising therapeutic target to eradicate CSCs. IgG (Invitrogen, Molecular Probes). Cell surface GPNMB proteins In this study, we investigated whether GPNMB, which induces were detected without membrane permeabilization. For IHC, tumorigenesis and EMT in mammary epithelial cells, affects bound antibodies were detected using ImmPRESS Reagent Kit acquisition of CSC-like properties in breast cancer cells. Our Peroxidase Anti-Goat IgG (Vector Laboratories). findings propose a novel model in which cell surface expression – of GPNMB induces stem cell like properties through hemITAM in Animal experiments dormant breast cancer cells. In other words, cell surface expression Six-week-old female NOD-SCID mice (CLEA) were subcuta- of GPNMB could be a desirable marker and therapeutic target of neously injected with 5 106 Hs578T cells. Four weeks later, the – breast cancer cells with stem cell like properties. mice were sacrificed and fresh tumors were obtained for RNA isolation or IHC staining as described above. 6 Materials and Methods For tumor formation using 4T1 cells, 1 10 4T1 cells were injected subcutaneously into 6-week-old female Balb/c mice Cell lines and cell culture (CLEA). After 3–4 weeks, the mice were sacrificed, and the tumors Breast cancer cell lines BT-474, Hs578T, MDA-MD-468, and were minced and dissociated under incubation conditions at 4T1 were obtained from ATCC (14). In the two-dimensional (2D) 37C in 1 mg/mL collagenase (Wako) for 2 hours, 0.25% trypsin monolayer cultures, cells were cultured in DMEM (Invitrogen) (Sigma) for 5 minutes, and 0.1 mg/mL DNase I (Roche) and supplemented with 10% FBS (Gibco), 100 U/mL penicillin G, and 5 mg/mL Dispase (Gibco) for 5 minutes. After centrifugation, 0.1 mg/mL streptomycin sulfate (Wako). In the 3D sphere cul- single cells were obtained and the cell number was counted tures, cells were cultured in DMEM/F12 (1:1) medium (Invitro- to perform FACS analysis and allograft transplantation for sec- gen) with 2% B-27 supplement (Invitrogen), 2 ng/mL bFGF ondary tumor formation. After 33 days, the mice were sacrificed, (Wako), 2 ng/mL EGF (Sigma), 100 U/mL penicillin G, and and the secondary tumors were collected. The tumor weight was 0.1 mg/mL streptomycin sulfate (Wako) in ultralow attachment measured and the volumes were approximated using the follow- culture dishes (Corning) or poly(2-hydroxyethyl methacrylate) ing formula: volume ¼ 0.5 a b2, where a and b are the lengths (Poly-HEMA; Sigma)-coated dishes. Hs578T cells were cultured in of the major and minor axes, respectively. m the presence of 10 g/mL insulin in both the 2D and 3D culture All the animal experiments were performed with the approval conditions. NMuMG-mock, NMuMG-GPNMB, and NMuMG- of the Animal Ethics Committee of the University of Tsukuba GPNMB (YF) cells were established and maintained as described (Ibaraki, Japan) and in accordance with the university's animal previously (14). Mycoplasma detection was performed using a experiment guidelines and the provisions of the Declaration of Mycoplasma Detection Set (Takara) for all the cell lines. Helsinki in 1995.

RNA interference FACS analysis 4T1 cells were transfected with 40 nmol/L of siRNA directed Two-dimensional–cultured cells were treated with trypsin (Sig- against GPNMB or control siRNA (Invitrogen) using Lipofecta- ma), and 3D-cultured spheres were dissociated using Accutase mine 3000 (Invitrogen) following the manufacturer's recommen- solution (Gibco). Single cells were incubated with anti-GPNMB dations. Stealth siRNAs (Invitrogen) were purchased as follows: antibody (AF2550; R&D Systems), and then with Alexa 488– siGpnmb #1 (MSS234870) and siGpnmb #2 (MSS294588). labeled donkey anti-goat IgG (Invitrogen, Molecular Probes) on Control siRNA was purchased from Invitrogen (Stealth RNAi ice for 30 minutes. The samples were analyzed using BD FACSAria Negative Universal Control Medium). (BD Biosciences) and BD FACSDiva software (100 mm Sorp AriaII 5B 3R 3V 5YG; BD Biosciences). We set the gating to collect cell Reverse transcription and qRT-PCR surface-GPNMBlow and -GPNMBhigh cells using the cells incubat- Total RNA was isolated using Isogen II (Nippon Gene). The ed with Alexa 488–labeled donkey anti-goat IgG only as the NucleoSpin RNA XS Kit (Macherey-Nagel) was used for extraction negative control. of total RNA from small samples after cell sorting. Reverse transcription was performed with High Capacity RNA-to-cDNA Master Mix (Applied Biosystems). mRNA levels were measured by Extreme limiting dilution analysis and sphere formation assay qPCR with gene-specific primers (Supplementary Table S1) using Extreme limiting dilution analysis (ELDA) was performed as SYBR Green I qPCR Master Mix (Applied Biosystems) on the ABI described previously (22). In brief, the breast cancer cells cultured low 7500 Fast Sequence Detection System. All samples were run in in 2D or 3D culture conditions and the cell surface-GPNMB high triplicate in each experiment. and -GPNMB subpopulations from 3D-cultured spheres were seeded in a series of numbers from 200 cells/well to 1 cell/well in m Western blot analysis 200- L sphere culture medium and cultured for 14 days. The Cells were suspended in 62.5 mmol/L Tris-HCl (pH 6.8) and number of wells containing spheres for each seeding cell number solubilized in SDS sample buffer [10% glycerol, 5% 2-mercap- was counted and then analyzed using online ELDA software toethanol, 2% SDS, and 62.5 mmol/L Tris-HCl (pH 6.8)]. Western (http://bioinf.wehi.edu.au/software/elda). blot analysis was performed as described previously (14). Statistical analysis Immunofluorescence and IHC staining Quantitative data were expressed as mean SEM. Statistical Immunofluorescence and IHC staining methods used were analyses were performed using the t test or one-way ANOVA with described previously (14, 21). Anti-GPNMB antibody (AF2550; the Tukey multiple comparison test using GraphPad Prism 7 R&D Systems) was used as the primary antibody, and cells were software. P < 0.05 was considered significant.

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Results the 2D-cultured cells (Fig. 1A–D; Supplementary Fig. S1A– GPNMB, CSC genes, EMT-TF genes, and mesenchymal marker S1D). We also examined CD24 mRNA expression because genes are enriched in 3D cultures BCSCs are often characterized by CD44high/CD24low popula- We enriched breast cancer stem cells (BCSC) using the 3D tions in breast cancer cells (24, 25). The expression levels of sphere culture method, which is used to examine the anchor- CD44 mRNA were higher and those of CD24 mRNA were lower age-independent growth potential (23). Three different human in the 3D-cultured cells than in the 2D-cultured cells (Fig. 1A– breast cancer cell lines, BT-474 (luminal type), Hs578T (basal D, two panels from the right). In addition, we examined the type), and MDA-MB-468 (basal type), and a mouse breast sphere-forming frequencies of the 2D- and 3D-cultured Hs578T cancer cell line, 4T1, were cultured in the 2D or 3D culture cells by ELDA (22). ELDA provides an estimate of sphere- conditions and compared for the expression levels of GPNMB forming frequency using one-sided confidence intervals and mRNA together with those of known CSC genes such as SOX2, statistical analysis. The 3D cultures yielded significantly higher NANOG, OCT4, CD44, CD133,andFOXO3.AllofthesemRNA sphere-forming frequency than did the 2D cultures (Supple- levels were significantly higher in the 3D-cultured cells than in mentary Fig. S1E).

Figure 1. Expression levels of GPNMB and CSC genes in 2D or 3D culture conditions. A–D, mRNA expression levels of GPNMB, SOX2, NANOG, OCT4, CD44, and CD24 in 2D- or 3D-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 to b-actin levels. , P < 0.01; , P < 0.001; t test. Data are representative of three independent replicates.

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GPNMB Induces Stemness in Dormant Breast Cancer Cells

Furthermore, we evaluated the expression of EMT-TF genes marker genes including CDH1, CDH2, ﬁbronectin,andvimentin such as SNAIL, SLUG,andZEB1 in BT-474, Hs578T, MDA-MB- in Hs578T and 4T1 as well. An epithelial marker, Cdh1,was 468, and 4T1 cells. The expression levels of these EMT-TF detected only in 4T1 cells and downregulated in the 3D cul- mRNAs were also enhanced in the 3D-cultured cells as com- tures. On the other hand, mesenchymal marker genes were pared with the levels in the 2D-cultured cells (Fig. 2A–D). enriched in the 3D-cultured both Hs578T and 4T1 cells (Sup- We also tested the expression of epithelial and mesenchymal plementary Fig. S1F and S1G).

Figure 2. Expression levels of EMT-TF genes in 2D or 3D culture conditions. A–D, mRNA expression levels of SLUG, SNAIL,andZEB1 in 2D- or 3D-cultured BT-474 (A), Hs578T (B), MDA-MD-468 (C), and 4T1 (D) cells were analyzed by means of qPCR (mean SEM; n ¼ 3). The results were normalized to b-actin levels. , P < 0.05; , P < 0.01; , P < 0.001; t test. Data are representative of three independent replicates.

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These results suggest that the 3D cultures can enrich the cells GPNMB is critical for induction of CSC-like properties in the with CSC-like properties in both human and mouse breast cancer 3D-cultured breast cancer cells. cell lines and that mRNA levels of GPNMB correlate with those of CSC genes, EMT-TF genes, and mesenchymal marker genes in this Correlation between growth arrest and expressions of GPNMB culture condition. and CSC genes and cell surface GPNMB protein exposure To examine the importance of GPNMB in the induction of We next evaluated the proliferative states of the cells in CSC genes and EMT-TF genes, we knocked down Gpnmb and different culture conditions and in in vivo tumorigenic condi- examined the expression levels of these genes in the 2D- and tion because one of the characteristics of CSCs is their slow 3D-cultured 4T1 cells. Knockdown of Gpnmb signiﬁcantly sup- proliferation or dormancy. MKI67-positive and -negative pressed the induction of CSC genes such as Sox2, Nanog,and Hs578T cell numbers were counted in the 2D and 3D cultures CD44, and of EMT-TF genes such as Snail, Slug,andZeb1,inthe by using immunoﬂuorescent staining and in xenograft tumors 3D-cultured 4T1 cells (Supplementary Fig. S2A and S2B). by using IHC staining. The MKI67-positive and -negative ratios Moreover, knockdown of Gpnmb reduced the sphere-forming in each condition are shown in Fig. 3A. More than 97% of the frequency of 4T1 cells (Supplementary Fig. S2C). Therefore, 2D-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 after immunofluorescent staining of 2D- or 3D-cultured Hs578T cells. IHC staining was used for counting MKI67-positive and -negative cells in Hs578T xenograft tumors. mRNA expression levels of MKI67 (B), SOX2,andGPNMB (C) in 2D- or 3D-cultured Hs578T and Hs578T tumors were analyzed by means of qPCR (mean SEM; n ¼ 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; n ¼ 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 are representative 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)or3D(G) cultures. Data are representative of more than three independent replicates.

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GPNMB Induces Stemness in Dormant Breast Cancer Cells

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 in 3D 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 are representative of three independent replicates. C–E, mRNA expression levels of GPNMB, SOX2, NANOG (C) SNAIL, SLUG (D), and MKI67 and TPX2 (E) in 2D-cultured Hs578T 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). 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; n.d., not detected. Data are representative of three independent replicates.

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the 3D-cultured spheres and nearly 50% of the xenograft suggest that growth-arrested conditions such as those after tumors were MKI67-negative. In addition, abundance of MKI67 serum starvation for confluent 2D cultures or 3D cultures can mRNA was significantly higher in the 2D-cultured cells than in activate the sorting of GPNMB protein for its exposure on the the 3D-cultured spheres or xenograft tumors (Fig. 3B). Inter- cell surface together with enhanced GPNMB transcription and estingly, SOX2 and GPNMB mRNA expression was enriched in CSC genes' induction. the spheres and tumors, and highly correlated with the MKI67- negative cell ratios, suggesting that dormant cells may have GPNMB is exposed on the surface of dormant breast cancer higher expression levels of SOX2 and GPNMB (Fig.3C).To stem-like cells in 3D-cultured spheres confirm whether growth arrest affects the expression of CSC To characterize the cell surface GPNMB-positive breast cancer genes and GPNMB, we induced growth arrest by serum star- cells, we isolated cell surface-GPNMBlow and -GPNMBhigh cell vation for 48 hours under subconfluent or confluent 2D culture populations from the 3D-cultured Hs578T spheres by FACS, conditions. Quantitative data obtained by qPCR showed that and the sphere-forming frequencies were compared in these the mRNA expression levels of CSC genes SOX2 and NANOG as two populations by use of ELDA together with cell proliferation well as GPNMB were increased in serum-free growth-arrested and CSC genes expression by qPCR (Fig. 4A). ELDA determined conditions, especially under confluent cell density (Fig. 3D). that approximately 1 in 76 cell surface-GPNMBhigh cells har- On the other hand, GPNMB protein was relatively abundant bored sphere-forming stem cell potential, while the cell surface- even in the 2D-cultured Hs578T cells without starvation GPNMBlow cells had much less frequency (about 1 in 1,955; (Fig. 3E; Supplementary Fig. S3A) when compared with the Fig. 4B). These results indicate significant differences in sphere- significantly higher induction of GPNMB mRNA in the 3D forming frequencies between the populations of the cell sur- cultures than in the 2D cultures (Fig. 1B, left). However, flow face-GPNMBlow and -GPNMBhigh cells. We further compared cytometry and immunofluorescent staining with/without per- the mRNA abundance of GPNMB and CSC genes SOX2 and meabilization revealed that less than 1% of the 2D-cultured NANOG in the 2D-cultured cells and in the cell surface- Hs578T cells had only low levels of cell surface GPNMB (Fig. GPNMBlow and -GPNMBhigh cells of 3D-cultured spheres. Both 3F), whereas nearly 10% of the cells were cell surface GPNMB- the cell surface-GPNMBlow and cell surface-GPNMBhigh cells positive in the 3D-cultured Hs578T cells (Fig. 3G; Supplemen- had higher expression levels of GPNMB, SOX2,andNANOG tary Fig. S3B). Similar regulation of GPNMB mRNA expression than did the 2D-cultured cells. Obviously, the cell surface- and cell surface protein exposure were confirmed with other GPNMBhigh population had much higher SOX2 and NANOG breast cancer cell lines, BT-474 and MDA-MB-468. These results than 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 cell surface-GPNMBlow and -GPNMBhigh cells in 3D-cultured 4T1 spheres. n ¼ 8 for each point. Data are representative of two independent replicates. B–D, mRNA expression 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 of 3D-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 Induces Stemness in Dormant Breast Cancer Cells

GPNMB mRNA expression levels did not differ significantly potential, while the cell surface-GPNMBlow cells had less fre- between the cell surface-GPNMBlow and -GPNMBhigh cells quency(Fig.5A).Next,wecomparedthemRNAabundanceof (Fig. 4C). Moreover, the expression levels of EMT-TF genes Gpnmb, Sox2,andNanog in the 2D-cultured cells and in the cell SNAIL and SLUG, EMT markers CDH2, fibronectin,andvimentin, surface-GPNMBlow and -GPNMBhigh cells of 3D-cultured and the proliferation marker genes MKI67 and TPX2 (26) spheres. The expression levels of Gpnmb, Sox2,andNanog in were examined subsequently. The expression levels of SNAIL the cell surface-GPNMBhigh cells were significantly higher than and SLUG (Fig. 4D) as well as of CDH2, fibronectin,andvimentin those in the 2D-cultured cells or the cell surface-GPNMBlow (Supplementary Fig. S4A) were highly correlated with those cells (Fig. 5B). Moreover, the cell surface-GPNMBhigh cells of SOX2 and NANOG. In contrast, the 2D-cultured cells had showed enhanced expression levels of Snail, Slug (Fig. 5C), the highest expression levels of proliferation marker genes, fibronectin,andvimentin (Supplementary Fig. S4B, middle while the cell surface-GPNMBhigh cells showed no detectable and right), whereas they showed the lowest levels of Mki67 levels of MKI67 or TPX2 (Fig. 4E). (Fig. 5D) and Cdh1 (Supplementary Fig. S4B, left). Taken In addition, we confirmed a similar phenomenon in a together, these results suggest that the cell surface-GPNMBhigh mouse breast cancer cell line 4T1. ELDA results showed that cells have the properties of dormant CSCs in the 3D-clutured the cell surface-GPNMBhigh cells had higher sphere-forming both human and mouse breast cancer cell lines.

Figure 6. GPNMB is exposed on cell surface of dormant cancer stem–like cells derived from breast tumors. A, ELDA was performed to determine sphere-forming frequencies of cell surface-GPNMBlow and -GPNMBhigh cells in 4T1 allograft tumors. n ¼ 8 for each point. Data are representative of two independent replicates. mRNA expression levels of Gpnmb, Sox2, Nanog (B), Snail, Slug (C), and Mki67 (D) in bulk tumor cells (indicated as "Tumor") and in cell surface-GPNMBlow and -GPNMBhigh cells of 4T1 allograft tumors were analyzed by means of qPCR (mean SEM; n ¼ 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. E and F, Secondary tumor-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). n ¼ 5. G and H, Cell surface GPNMB was 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).

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GPNMB is exposed on the surface of dormant breast cancer lected isolated single cells from the tumors and sorted cell surface- stem-like cells in allograft tumors and enhances tumorigenicity GPNMBlow and -GPNMBhigh cells by FACS. ELDA showed that the We further examined the characteristics of the cell surface- cell surface-GPNMBhigh cells harbored higher sphere-forming GPNMBlow and -GPNMBhigh cells using a 4T1 allograft tumor frequency (about 1 stem cell in 29 cells), while the cell surface- model. We compared the mRNA abundance of Gpnmb, Sox2, and GPNMBlow cells had much less sphere-forming frequency (about Mki67 in the 2D or 3D-cultured 4T1 cells and 4T1 tumors. The 4T1 1 in 1,065; Fig. 6A). Moreover, the cell surface-GPNMBhigh tumor tumors had lower expression levels of Gpnmb and Sox2 than did cells showed the highest expression levels of Gpnmb, Sox2, Nanog the 3D-cultured spheres, but those levels were still more enriched (Fig. 6B), Snail, and Slug (Fig. 6C), and the lowest expression level than those of the 2D-cultured cells (Supplementary Fig. S5A). of Mki67 mRNA (Fig. 6D). These results indicated that like Mki67 expression levels were lower in the 3D-cultured cells or 3D-cultured spheres, the tumors grown in vivo are also composed tumors than in the 2D-cultured cells, similar to Hs578T tumors of cell surface-GPNMBlow and -GPNMBhigh cells and that the cell (Supplementary Fig. S5B). After harvesting 4T1 tumors, we col- surface-GPNMBhigh cells have dormant CSC-like properties.

Gpnmb Sox2 Nanog

1,500 6 70 65 1,000 60 500 4 55 50 10 15 8 n.s. 2 6 b -Actin mRNA n.s. 10 4 n.s. 2 n.d. 5 0 0 Sox2/ 0 Gpnmb/ b -Actin mRNA

2D 3D 2D 3D 2D 3D 2D 3D 2D 3D 2D 3D Noanog/ b -Actin mRNA 2D 3D 2D 3D 2D 3D

(Fold expression vs. mock 2D) Mock GPNMB GPNMB (YF) Mock GPNMB GPNMB (YF) Mock GPNMB GPNMB (YF) (Fold expression vs. mock 2D) (Fold expression vs. GPNMB 2D)

Surface GPNMBhigh High CSC genes Sphere/Tumor High EMT-TF genes Dormancy High Sphere-forming frequency High tumorigenicity

Surface GPNMBlow GPNMB Low CSC genes Low EMT-TF genes Rapid proliferation Cell surface GPNMBhigh cells Low Sphere-forming frequency Low tumorigenicity

Cell surface GPNMBlow cells

Figure 7. The tyrosine residue in hemITAM of GPNMB is essential for induction of CSC genes. A, mRNA expression levels of Gpnmb, Sox2,andNanog 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 results were 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. Cell surface GPNMB induces the expression of CSC genes and EMT-TF genes in dormant breast cancer cells and contributes to the tumorigenicity.

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GPNMB Induces Stemness in Dormant Breast Cancer Cells

Furthermore, we tested the secondary tumor growth of the cell Fig. S5A). Taken together, these results indicate that the 3D culture surface-GPNMBlow and -GPNMBhigh 4T1 tumor cells. Injected cell method is a useful in vitro system to examine CSC-like properties. surface-GPNMBhigh cells generated secondary tumors with a CSCs are found in acute myeloid leukemia (36) and solid higher incidence rate (103, 3/5; 104, 5/5) than that of the cell tumors including those of the breast (24), brain (37), prostate surface-GPNMBlow cells (103, 1/5; 104, 1/5; Fig. 6E and F; (38), colon (39), pancreas (40), and lung (41). Because BCSCs þ Supplementary Fig. S5C and S5D). These results clearly indicated were isolated as cells with the markers of CD44 /CD24 /low/Lin þ that the cell surface-GPNMBhigh cells have higher secondary (24), CD44 /CD24 is frequently used as a BCSC marker (25). tumor-forming frequency. In addition, further analysis of the Other surface markers, such as CD133, CD49f, and CD61, have tumors generated by the cell surface-GPNMBlow cells indicated also been reported (42–45). Whereas GPNMB was located mainly that these tumors were also composed of a comparable ratio of cell on lysosome or endosome membranes in 2D-cultured cells, it was surface-GPNMBhigh and -GPNMBlow cells, with the tumor made exposed on the cell surface of dormant BCSCs and induced stem from the cell surface-GPNMBhigh cells, suggesting that the plas- cell-like properties in 3D-cultured spheres and in vivo tumors ticity of cancer cells generate cell surface-GPNMBhigh cells from (Figs. 3F and G, 4B–E, 5, and 6). Our results indicate that surface the cell surface-GPNMBlow cells at low frequency and causes the expression of GPNMB could be a novel indicator of dormant tumorigenic potential (Fig. 6G and H). BCSCs. However, the frequency with which stem cells were enriched with cell surface-GPNMBhigh cells in the 3D-cultured spheres or tumors was not 100% (Figs. 4B, 5A, and 6A). Therefore, The tyrosine residue in GPNMB hemITAM is essential for investigation of possible and more suitable combinations of induction of CSC genes BCSC markers might be required for further characterization of To elucidate the molecular mechanism of GPNMB in genera- breast cancer stem-like cells. tion of CSC-like properties, we examined the involvement of the Although we have not elucidated the mechanism whereby tyrosine residue in hemITAM, because we have previously dem- GPNMB is exposed on the surface of dormant cells, we have onstrated that Tyr529 is essential for GPNMB-inducible EMT and here shown that growth-arrested conditions, such as serum tumorigenesis. We used NMuMG-mock, NMuMG-GPNMB, and starvation or 3D culture conditions, induced transcriptional NMuMG-GPNMB(YF) cell lines, in which Tyr529 is replaced by a activation of GPNMB and total GPNMB protein (Fig. 3D–G; phenylalanine (14). Exogenous Gpnmb mRNA was highly Supplementary Fig. S3). In addition, GPNMB is exposed on the expressed in both NMuMG-GPNMB and NMuMG-GPNMB(YF) cell surface only in MKI67-negative growth-arrested cells cells cultured in the 2D and 3D culture conditions (Fig. 7A, left; (Figs. 4E, 5D, and 6D). Moreover, cell growth analyses shown Supplementary Table S2, top), but the expression of the CSC in Fig. 3A indicated that the 2D cultures mostly composed of genes Sox2 and Nanog was induced in NMuMG-GPNMB cells monotonous MKI67-positive proliferating cells. On the other cultured in the 3D culture condition only. On the other hand, hand, the 3D cultures and in vivo tumors contain both growth- GPNMB(YF) cells failed to induce the expression of Sox2 and arrested cells and proliferating cells. This divergence in cell Nanog mRNA, even in the 3D culture condition (Fig. 7A, middle proliferation status is thought to make the critical difference in and right; Supplementary Table S2, middle and bottom). These 2D-culturedcellsand3D-culturedspheresorin vivo tumors. results indicate that GPNMB function mediated by the tyrosine Qian and colleagues have reported that inhibition of the ERK residue in hemITAM is essential for the induction of CSC genes in signaling pathway enhances GPNMB protein expression in the 3D cultures. several melanoma cell lines that have NRAS or BRAF mutations (46). Taken together, these findings suggest that growth inhi- Discussion bition may enhance GPNMB transcriptional activation and In this article, we have reported a novel function of GPNMB, protein sorting to the cell surface, and leads to maintenance which is exposed on the surface of growth-arrested breast cancer of CSC-like phenotypes. cells and induces stem cell–like properties through hemITAM GPNMB contains hemITAM (YxxI) and a dileucine motif (D/ (Fig. 7B). ExxLL) in its cytoplasmic tail. These motifs are frequently found in We previously demonstrated that GPNMB can induce EMT in transmembrane proteins and function as sorting signals associ- NMuMG cells (14). The correlation between EMT and the acqui- ated with endocytosis or endosomal/lysosomal membrane traf- sition of stem cell-like properties has been proven in breast ficking (47). Our previous study demonstrated that hemITAM is epithelial cells (15), and EMT-TFs, including TWIST, SLUG, essential for inducing EMT and tumorigenesis via phosphoryla- SNAIL, ZEB1, and ZEB2, have been shown to confer the char- tion of the tyrosine residue by SRC kinase (14). We have here acteristics of CSCs (15, 27, 28). In addition, Chen and colleagues further shown that it is involved in the induction of CSC genes showed that 3D-cultured cells had higher expression of the EMT- in the 3D cultures (Fig. 7A). The loss tumorigenicity made TF genes SNAIL and TWIST than did 2D-cultured cells in head and it difficult to analyze the gene expression levels of mutant neck squamous cell carcinoma cell lines (29). The 3D culture hemITAM-expressing tumors in vivo. Moreover, Lin and collea- system is thought to mimic the cellular dynamics in the body gues reported that cell surface GPNMB forms a heterodimer with better than the 2D culture system (30–32) and is used to enrich EGFR and that the tyrosine residue in hemITAM is phosphory- cells with stem cell potential in cancers of the breast (33, 34), brain lated upon heparin-binding EGF (HB-EGF) stimulation. Subse- (35), and head and neck (29). In our 3D cultures, the spheres had quently, the BRK– or LRRK2–LINK-A complex binds to GPNMB enhanced expression levels of CSC genes as well as of EMT-TF and stabilizes normoxic HIF1a. They also showed that phosphor- genes and altered those of EMT marker genes (Figs. 1, 2, 4C and ylation of GPNMB is detected in TNBC and correlates with the D, 5B and C, and 7A; Supplementary Fig. S1A–S1D, S1F, S1G, and metastatic status of patients with breast cancer (48). We have also S4). Importantly, similar results were observed in the xenograft shown that EGFR and FGFR1 were enriched in the 3D-cultured and allograft tumors (Figs. 3C, 6B and C; Supplementary Hs578T and 4T1 cells (Supplementary Fig. S6A and S6B).

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Chen et al.

Therefore, it is worthwhile examining the cross-talk between Disclosure of Potential Conflicts of Interest tyrosine receptor kinase signaling and GPNMB in detail. These No potential conflicts of interest were disclosed. results together with our current observations suggest that both cell surface expression of GPNMB and phosphorylation of the Authors' Contributions tyrosine residue in hemITAM are essential for the GPNMB-induc- Conception and design: C. Chen, Y. Okita, M. Kato ible tumorigenic and metastatic potential, at least partly owing to Development of methodology: C. Chen, Y. Okita, F. Abe, M. Kato the generation of CSC-like properties. Acquisition of data (provided animals, acquired and managed patients, GPNMB is known to have a soluble form shed by ADAM10 provided facilities, etc.): C. Chen, Y. Okita, Y. Watanabe, F. Abe, M.A. Fikry, (49), and it could be detected in patients' blood (8). Therefore, Y. Ichikawa, A. Shibuya, M. Kato shedding of GPNMB is thought to happen on the cell surface. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C. Chen, Y. Okita, F. Abe, M. Kato So far we know of no relationship among GPNMB cell surface Writing, review, and/or revision of the manuscript: C. Chen, Y. Okita, localization, hemITAM tyrosine phosphorylation, and shed- H. Suzuki, M. Kato ding of its extracellular domain and it would be interesting to Administrative, technical, or material support (i.e., reporting or organizing study these points as a serial phenomenon to understand the data, constructing databases): H. Suzuki physiologic and pathologic functions of GPNMB. Study supervision: Y. Okita, M. Kato CDX-011 (glembatumumab vedotin) is an antibody–drug conjugate that contains CR011, a human mAb against GPNMB, Acknowledgments and conjugates with a cytotoxic agent (MMAE). CDX-011 has We thank F. Miyamasu (Medical English Communication Center, University been developed for the treatment of GPNMB-expressing cancers, of Tsukuba, Ibaraki, Japan) for proofreading the manuscript. This research was and clinical studies of CDX-011 against patients with breast cancer supported by JSPS Grant Numbers JP15K19070, JP17K14981, and JP18H02676 and melanoma were conducted (12, 13, 50). We have shown that and grants from the Naito Foundation and the Uehara Memorial Foundation. the cell surface-GPNMBhigh cells have higher stem cell–like prop- The costs of publication of this article were defrayed in part by the payment of erties than do the cell surface-GPNMBlow cells (Fig. 7B). Thus, our fi page charges. This article must therefore be hereby marked advertisement in novel ndings lead us to propose that targeting GPNMB, which is accordance with 18 U.S.C. Section 1734 solely to indicate this fact. exposed on the surface of dormant cancer cells, has better feasi- bility to kill CSCs and might result in a highly efficient cancer Received February 23, 2018; revised July 31, 2018; accepted September 12, treatment. 2018; published first September 17, 2018.

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www.aacrjournals.org Cancer Res; 78(22) November 15, 2018 6435

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

Chen Chen, Yukari Okita, Yukihide Watanabe, et al.

Cancer Res 2018;78:6424-6435. Published OnlineFirst September 17, 2018.

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