Stem Cell Antigen-1 Enhances Tumorigenicity by Disruption of Growth Differentiation Factor-10 (GDF10)–Dependent TGF-Β Signaling
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Stem cell antigen-1 enhances tumorigenicity by disruption of growth differentiation factor-10 (GDF10)–dependent TGF-β signaling Geeta Upadhyaya, Yuzhi Yina, Hongyan Yuana, Xin Lib, Rik Derynckc, and Robert I. Glazera,1 aDepartment of Oncology, Lombardi Comprehensive Cancer Center and bDepartment of Biostatistics, Bioinformatics and Biomathematics, Georgetown University Medical Center, Washington, DC 20007; and cDepartment of Cell and Tissue Biology, University of California, San Francisco, CA 94143 Edited by Joan S. Brugge, Harvard Medical School, Boston, MA, and approved April 1, 2011 (received for review March 2, 2011) Stem cell antigen (Sca)-1/Ly6A, a glycerophosphatidylinositol- mary cultures derived from mammary tumors showed a fourfold linked surface protein, was found to be associated with murine higher level of Sca-1-expressing cells than cultures from normal stem cell– and progenitor cell–enriched populations, and also has mammary gland (Fig. S1D). To explore the role of Sca-1, we been linked to the capacity of tumor-initiating cells. Despite these established a tumor cell population, designated 34T, from an +/EGFP interesting associations, this protein’s functional role in these pro- adenocarcinoma induced in Sca-1 mice. We used these 34T cesses remains largely unknown. To identify the mechanism un- cells, which were >80% positive for Sca-1 (Fig. S1E), to examine derlying the protein’s possible role in mammary tumorigenesis, the signaling pathways regulated by Sca-1. Sca-1 expression was examined in Sca-1+/EGFP mice during carcino- genesis. Mammary tumor cells derived from these mice readily Attenuation of Sca-1 Expression Reduces Cell Growth and engrafted in syngeneic mice, and tumor growth was markedly Tumorigenicity. To examine whether Sca-1 plays a tumorigenic inhibited on down-regulation of Sca-1 expression. The latter effect role, we transduced 34T cells with a lentivirus expressing either was associated with significantly elevated expression of the TGF-β GFP shRNA as a control or Sca-1 shRNA to generate a stable cell ligand growth differentiation factor-10 (GDF10), which was found population with reduced Sca-1 expression (Fig. S2). 34T cells ex- to selectively activate TGF-β receptor (TβRI/II)-dependent Smad3 pressing Sca-1 shRNA D8 (D8 cells) exhibited a 90% reduction of Sca-1 protein expression compared with 34T/GFP shRNA CELL BIOLOGY phosphorylation. Overexpression of GDF10 attenuated tumor for- cells (34T cells) (Fig. 1 A and B). 34T cells grew as clusters with mation; conversely, silencing of GDF10 expression reversed these a spheroid morphology, whereas D8 cells lost the spheroid mor- effects. Sca-1 attenuated GDF10-dependent TGF-β signaling by dis- β β phology (Fig. 1C) and displayed reduced anchorage-independent rupting the heterodimerization of T RI and T RII receptors. These colony formation (Fig. 1D). To test the tumorigenic potential of findings suggest a new functional role for Sca-1 in maintaining β Sca-1, we engrafted syngeneic mice with 34T and D8 cells. D8 tumorigenicity, in part by acting as a potent suppressor of TGF- cells either failed to develop tumors or grew as small tumors, signaling. whereas 34T cells formed large tumors after 25 d (Fig. 1E). tem cell antigen (Sca)-1 is a member of the Ly6A superfamily Sca-1 Controls Cell Behavior and Tumorigenicity Through Inhibition of Sof glycerophosphatidylinositol (GPI)-anchored membrane GDF10 Expression. To determine whether the Sca-1–associated proteins (1), which is associated with murine stem and pro- changes in growth is related to secreted growth inhibitory factors, genitor cell populations in several tissues. In the mammary we assessed colony formation in the presence of conditioned gland, Sca-1–positive cells are able to reconstitute the cleared fat media from 34T and D8 cells. Conditioned medium from control pad (2, 3) and give rise to alveolar and ductal structures (4). In 34T cells partially restored the growth of D8 cells, whereas the addition to Sca-1’s normal role in stem cell self-renewal, Sca-1 medium from D8 cells inhibited the growth of 34T cells (Fig.1F). expression is elevated in malignant tissues, such as retino- To further assess the phenotypic changes associated with Sca-1 blastomas (5), prostate tumors (6), mammary tumors (7, 8) and expression, we carried out gene microarray analyses in both cell chronic myeloid leukemia (9), which generally reflects a more populations. Among the 57 expressed genes that exhibited a aggressive phenotype (10). Despite these associations, the role of threefold or greater change in D8 cells, the TGF-β ligand Sca-1 in these processes remains largely unknown. To address GDF10 exhibited the largest change (Fig. S3 and Table S1). this question, we used Sca-1+/EGFP mice, in which EGFP is under Because several TGF-β ligands are known to inhibit cell pro- the control of the Sca-1 locus (11), to study mammary tumori- liferation, the expression of additional TGF-β ligands was de- genesis. We found that Sca-1 suppresses TGF-β signaling by termined in 34T and D8 cells (Fig. 1G). Only GDF10 expression inhibiting expression of the TGF-β family ligand GDF10 and was significantly increased in D8 cells; no changes in TGF-β1, binding to the type I TGF-β receptor (TβRI), thereby inhibiting TGF-β2, and TGF-β3 expression were seen. ligand-induced TGF-β receptor complex formation and Smad3 We next evaluated the role of GDF10 in tumor cell growth by phosphorylation. This study is the first to demonstrate specific generating 34T cells that stably express GDF10 (Fig. 1H) and D8 functions for Sca-1 and GDF10 in tumorigenesis. cells that express a GDF10 shRNA and thus have reduced GDF10 expression (Fig. 1I). Increased GDF10 expression in Results 34T/GDF10 cells resulted in reduced colony formation, whereas Sca-1 Is Increased Early in Mammary Tumorigenesis. To explore the reducing GDF10 expression enhanced the colony formation of role of Sca-1 in tumorigenesis, we induced mammary tumors in heterozygous Sca-1+/EGFP mice (11) using medroxyprogesterone (MPA) and 7,12-dimethylbenz[a]anthracene (DMBA) (8, 12, 13). Author contributions: G.U., R.D., and R.I.G. designed research; Y.Y. and H.Y. developed Cultures of primary mammary epithelial cells exhibited enhanced the 34T cell line; X.L. performed the bioinformatic analysis; G.U. performed research; EGFP fluorescence coincident with increased Sca-1 expression G.U., R.D., and R.I.G. analyzed data; and G.U., R.D., and R.I.G. wrote the paper. immediately after treatment with MPA and DMBA ((Fig. S1 A The authors declare no conflict of interest. and B ), suggesting that the Sca-1 locus is activated at the onset This article is a PNAS Direct Submission. +/EGFP of carcinogenesis. Both Sca-1 and WT mice developed 1To whom correspondence should be addressed: E-mail: [email protected]. fi tumors within 2 mo of MPA/DMBA treatment, with no signi cant This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. difference in the incidence of tumor formation (Fig. S1C). Pri- 1073/pnas.1103441108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1103441108 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 Fig. 1. Silencing Sca-1 expression ABCD decreases colony formation and tu- 34T 34T 0.5 morigenic potential, and increases 0.45 GDF10 expression. 34T cells were 34T D8 0.4 transduced with a lentivirus ex- IgG 34T Sca-1 0.35 pressing either GFP shRNA as a con- 0.3 trol or a Sca-1 shRNA (Fig. S2). D8 D8 Actin D8 D8 0.25 cells expressing Sca-1 shRNA cells 0.2 Colony growth growth Colony Counts exhibited a 90% reduction in Sca-1 0.15 expression compared with control 0.1 34T cells, as assessed by FACS analy- 0.05 sis (A) or Western blot analysis 0 (B). (C) Down-regulation of Sca-1 Sca-1 34T D8 expression reduces spheroid cluster morphology. 34T cells grew as EF34T D8 G spheroid clusters, whereas D8 cells 1 2 1.2 34T 128 grew as a flattened monolayer with D8 64 contact-inhibited growth. (D) Silenc- 600 1 ) ing Sca-1 expression reduces colony 3 500 32 0.8 formation. Colony-formation assay 400 3 4 16 in D8 cells vs. 34T cells showed that 0.6 300 growth was reduced by 70% in D8 * 8 34T CM34T No CM cells vs. 34T cells. (E) Down-regulation 200 Colony growth 0.4 P <0.001 4 Tumor Volume (mm 5 6 of Sca-1 expression markedly re- 100 0.2 34T vs. D8 in levels mRNA duces tumorigenicity. 34T cells and 1 2 3 4 5 6 0 2 D8 cells were implanted s.c. at an 34T D8 D8 CM D8 0 inoculum of 100,000 cells into op- CM None 34T D8 1 1 2 3 posite flanks of eight syngeneic β β β TGF- TGF- TGF- C57BL/6 mice. Tumor formation was GDF10 monitored over 25 d, and tumor volume was calculated. Isografts HI J1.6 K from D8 cells exhibited little growth 140 1.2 1000 compared with isografts from 34T 1.4 120 1 cells (P < 0.001, two-tailed Student t 1.2 ) 100 3 test). (F) Conditioned medium (CM) 0.8 1 750 from 34T cells enhanced colony for- mRNA 80 0.6 0.8 mation of D8 cells, whereas condi- 60 0.6 500 GDF10 GDF10 mRNA GDF10 tioned medium from D8 cells 0.4 Colony growth 40 markedly reduced colony formation 0.4 0.2 Tumor Volume (mm of 34T cells. The bar graph shows 20 0.2 250 quantification of colony growth; the 0 0 0 D8 D8 34T number in each bar corresponds to 34T 0 the plate number in the left panel.