Stem Cell Antigen-1 Enhances Tumorigenicity by Disruption of Growth Differentiation Factor-10 (GDF10)–Dependent TGF-Β Signaling

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 carcinogenesis. 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 posite ﬂanks of eight syngeneic β 1 β 2 β 3 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. 34T 34T/GDF10 D8 D8/GDF10sh (G) Real-time qPCR analysis of TGF-β 34T/GDF10 34T/GDF10 D8/GDF10sh ligands. The fold increase in expres- D8/GDF10sh sion of each ligand in D8 cells relative to 34T cells is shown. D8 cells expressed >100-fold higher levels of GDF10 mRNA compared with 34T cells. (H) 34T/GFP shRNA cells stably expressing GDF10 (34T/GDF10) exhibit a >100-fold increase in GDF10 mRNA expression compared with control cells (34T). (I)D8cells transfected with a GDF10 shRNA (D8/GDF10sh) show 90% reduced GDF10 mRNA expression compared with cells expressing a control shRNA (D8). (J) Modu- lation of GDF10 expression results in changes in colony formation. Expression of GDF10 in 34T cells (34T/GDF10) reduced colony formation by 80%, whereas silencing GDF10 expression in D8 cells (D8/GDF10sh) increased colony formation. (K) GDF10 modulates tumorigenicity. Cells were implanted into eight syngeneic C57BL/6 mice, and tumor growth was assessed as in E. Tumor volume was reduced in isografts of 34T/GDF10 cells compared with tumors from 34T cells (P < 0.001, two-tailed Student t test). In contrast, D8/GDF10sh cells showed increased tumor volume compared with D8 cells (P < 0.001, two-tailed Student t test).

D8 cells (Fig. 1J and Fig. S4A). In addition, increasing GDF10 for Smad3 (TGF-β) and Smad1/5 (BMP) signaling (Fig. 2A). The expression reduced the spheroid morphology and restored the Smad3 reporter activity was strongly increased in D8 cells, in contact inhibition of 34T cells, whereas down-regulation of contrast to the low and unchanged Smad1/5 reporter activity, GDF10 expression had an opposite effect in D8 cells (Fig. S4B). demonstrating a correlation of decreased Sca-1 expression and We then evaluated the tumorigenic properties of these cells in increased GDF10 expression with increased Smad3 activity. In isograft experiments in syngeneic mice (Fig. 1K). 34T cells addition, GDF-10 overexpression in 34T cells increased TGF-β formed large tumors after 25 d (Fig. 1E), whereas 34T/GDF10 reporter activity, and silencing GDF10 expression in D8 cells cells formed considerably smaller tumors. Conversely, D8 cells produced the opposite effect (Fig. 2B). This outcome is consistent failed to grow as isografts (Fig. 1E), but decreasing GDF10 ex- with increased expression of the TGF-β target genes Smad6 (Fig. pression conferred tumorigenicity (Fig. 1K). Thus, Sca-1 regu- 2C) and PAI-1 (Fig. 2D) in 34T/GDF10 cells and decreased ex- lates tumorigenicity in part by repressing GDF10 expression. pression of these genes when GDF10 shRNA was introduced in D8 cells. These results indicate a correlation of GDF10 expression Sca-1 and GDF10 Regulate Signaling Through Smad3. GDF10 was and signaling with activation of Smad3 signaling. To verify this previously identified as a TGF-β ligand related to BMP-3 (14), notion, we examined the Smad3 activation in 34T and D8 cells although there is little information on its mechanism of action. (Fig. 2E). We found a much higher level of C-terminal Smad3 Among several TGF-β family ligands, TGF-βs and activins signal phosphorylation in the D8 cells than in the 34T cells, consistent primarily through activation of Smad2 and Smad3, whereas BMPs with the significant increase in GDF10 production. Furthermore, act through Smad1 and 5 (15, 16). To determine the effect of in- the phosphoSmad3 level in 34T cells was increased by GDF10 creased GDF10 expression in D8 cells, we compared the autocrine overexpression, and the high level of phosphoSmad3 in D8 cells Smad signaling in 34T and D8 cells using reporter assays specific was reduced on down-regulation of GDF10 expression. These

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1103441108 Upadhyay et al. Downloaded by guest on September 26, 2021 ABCDE

250 6 3 300 34T 34T/GDF10 D8 D8/GDF10sh 5 200 250 2 pSmad3 ) 3 ) 3 4 150 200 2 Smad2/3

3 RNA levels

150 m 1 100 pSmad1 2 β Reporter (x10 Smad6 mRNA levels 100 PAI1

Luc reporter (x10 50 1 1 Smad1/5 TGF- 50 0 0 0 0 D8 D8 Smad4 D8 D8 34T 34T 34T 34T D8 34T β TGF- BMP β-actin 34T/GDF10 34T/GDF10 D8/GDF10sh D8/GDF10sh 34T/GDF10 D8/GDF10sh

F Smad2/3 DAPI Merge Smad1/5 DAPI Merge Smad2/3 Smad1/5 45 * 40 35 * 30 25 20 15 10 D8 34T/GDF10 34T Nuclear labelingSmads of 5 * 0 D8 34T CELL BIOLOGY /GDF10sh D8 34T/GDF10 D8/GDF10sh

Fig. 2. Silencing Sca-1 expression activates GDF10-dependent signaling through Smad3. (A) D8 cells exhibit increased Smad3 (TGF-β), but not Smad1/5 (BMP) reporter activity. D8 cells expressed a >20-fold increase in Smad3 activity compared with 34T cells. (B) GDF10 expression increases Smad3 activity. Reporter activity was increased in GDF10-overexpressing 34T cells (34T/GDF10), whereas silencing GDF10 expression in D8 cells (D8/GDF10sh) reduced the activity. (C and D) Expression of the TGF-β target genes Smad6 (C) and PAI1 (D) is regulated by GDF10 expression. Both Smad6 and PAI1 expression were increased by overexpression of GDF10 in 34T cells and decreased by down-regulation of GDF10 expression in D8 cells. (E) GDF10 expression regulates Smad3 activation, as assessed by immunoblotting for phosphoSmad3 (pSmad3). The levels of Smads and pSmads were determined by Western blot analysis. pSmad3 was increased in 34T/ GDF10 cell, compared with 34T cells and was reduced in D8/GDF10sh cells compared with D8 cells. No difference in pSmad1/5 was observed. (F) GDF10 expression correlates with Smad2/3 nuclear localization. Fluorescence was measured by confocal microscopy using a Smad2/3 or Smad1/5 antibody and an Alexa Fluor 594-conjugated secondary antibody; nuclei were stained with DAPI and pseudo-colored in green. The merged image shows nuclear localization of Smad2/ 3 in 34T/GDF10 and D8 cells, but not in 34T and D8/GDF10sh cells. The bar graph quantiﬁes Smad2/3 and Smad1/5 nuclear localization. *P < 0.01, Student two- tailed t test. (Scale bar: 10 μm.)

results are correlated with the extent of nuclear localization of GDF10 used the same type I and type II receptors as TGF-β1 Smad2/3 and Smad1/5 in 34T, 34T/GDF10, D8 and D8/GDF10sh (i.e., TβRII and TβRI), we carried out [125I]TGF-β receptor cells (Fig. 2F). binding assays of intact cells (17–19) using unlabeled GDF10 as a competitor (Fig. 3E). [125I]TGF-β cross-linking visualized the GDF10 Activates Smad3 Signaling via TβRI and TβRII. To determine abundant betaglycan (type III TGF-β receptor), as well as the whether GDF10 directly activates Smad3 similar to TGF-β,we 70-kDa TβRII and 53-kDa TβRI. Unlabeled TGF-β1 and treated NMuMG cells with recombinant GDF10 or TGF-β1 (Fig. GDF10 were effective at competing with [125I]TGF-β binding to 3A). Both GDF10 and TGF-β1 induced the nuclear localization of betaglycan, TβRII, and TβRI, although TGF-β1 was more ef- Smad2/3 but not of Smad1/5, suggesting that they act through fective in competing than GDF10. We next compared the effects − − a common receptor–Smad pathway. Accordingly, GDF10 induced of GDF10 and TGF-β1 in WT versus Tgfbr1 / mouse embryo the expression of the TGF-β target genes Smad6 and PAI-1 (Fig. ﬁbroblasts (MEFs). Both had equal expression of ALK4 and 3B), consistent with our results using 34T or D8 cells with in- ALK7, whereas the latter speciﬁcally lacked the expression of − − creased or decreased GDF10 expression (Fig. 2 C and D). In ad- TβRI (Fig. 3F, Upper). Treatment of WT, but not Tgfbr1 / cells, dition, GDF10 induced Smad3 phosphorylation in 34T cells as with GDF10 resulted in a time-dependent increase in pSmad3 rapidly as TGF-β1, although at 30 min, TGF-β1 induced a higher (Fig. 3F, Lower), indicating that GDF10 signals through TβRI. In level of Smad3 activation than GDF10 (Fig. 3C). Adding GDF10 addition, GDF10 was unable to induce Smad3 activation in to D8 cells, which express high levels of GDF10, enhanced the MEFs with inactivated expression of TβRII (Fig. 3G), indicating constitutive activation of Smad3 only minimally; however, this was the requirement for TβRII. Taken together, our results indicate further enhanced in response to TGF-β1 (Fig. 3C). Thus, although that GDF10 acts through the same TβRII/TβRI receptor com- both GDF10 and TGF-β activate Smad3, TGF-β1 is more potent in plex as TGF-β to activate Smad3, and that increased GDF10 activating Smad3 compared with GDF10. expression as a result of down-regulation of Sca-1 expression As assessed by real-time qPCR, 34T and D8 cells expressed results in autocrine activation of TβRI/II signaling. the ALK4, TβRI/ALK5, and ALK7 type I receptors, which all are able to activate Smad2 and Smad3, as well as the TβRII, Sca-1 Associates with TβRI to Inhibit Smad3 Signaling. To evaluate ActRII, and BMPRII type II receptors (Fig. 3D). These recep- the direct effect of Sca-1 on ligand-induced Smad3 activation, we tors were expressed at similar levels with the exception of TβRII, treated WT and Sca-1–overexpressing NMuMG cells with which was increased twofold in D8 cells. To determine whether GDF10 or TGF-β1. Both ligands induced Smad3 activation in

Upadhyay et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 Fig. 3. GDF10 activates Smad2/3 signaling A Smad2/3 through the TGF-β pathway. (A)GDF10 Smad1/5 increases Smad3 nuclear localization. Treat- Smad2/3 DAPI Merge Smad1/5 DAPI Merge 40 * ment of NMuMG cells for 30 min with 50 ng/ 35 * mL of GDF10 increased nuclear localization Control 30 of Smad2/3. Treatment with 5 ng/mL of TGF- 25 β1 served as a positive control. The bar graph 20 quantitates nuclear Smad2/3 and Smad1/5. GDF10 (Scale bar: 10 μm.) (B) GDF10 induces Smad6 15 and PAI1 mRNA expression. NMuMG cells 10 β treated overnight with GDF10 and TGF- as TGF-β1 5 Nuclear labeling of Smads in A expressed increased levels of mRNAs for 0 the TGF-β target genes Smad6 and PAI1, as GDF10 - - + + - - determined by real-time qPCR. (C)GDF10 TGF-β1- - - - + + treatment activates Smad3. 34T and D8 cells were treated with either GDF10 or TGF-β1, BC D and pSmad3 levels were determined by Smad6 PAI1 2.0 34T D8 Western blot analysis. pSmad3 was increased 4.5 at 10 min in 34T cells to a greater extent 0 10 30 0 10 30 min 1.5

3.5 pSmad3 34T vs. in D8 than in D8 cells due to the higher basal level GDF10 1.0 in the latter cells; pSmad3 at 30 min was Smad2/3 β 2.5 levels equivalent in both cell lines. TGF- 1in- pSmad3 0.5 β creased pSmad3 to a greater extent than TGF- 1 RNA β levels mRNA 1.5 Smad2/3 m 0.0 GDF10 in both 34T and D8 cells. (D) TGF- 5 receptor expression in 34T and D8 cells. Re- β RII ALK4 ALK7 T ﬁ 0.5 ActRII BmpRII

ceptor mRNA was quanti ed by real-time - + - + GDF10 β RI/ALK qPCR. Only TβRII mRNA was signiﬁcantly T different in D8 and 34T cells (P < 0.05, two- tailed Student t test). (E) GDF10 competes E F Wild type Tgfbr1-/- G Wild type Tgfbr2-/- GDF10 1.5 with TGF-β1 for receptor binding. Mv1Lu TGF-β1 + + GDF10, min 0 10 30 0 10 30 [125I] TGF-β1 + + + + + + + + cells were surface-labeled with 5 ng/mL of pSmad3 125 β 1.0 [ I]TGF- 1 and competed with either 100 Smad3 ng/mL of unlabeled TGF-β1 or 100–200 ng/ 250 kDa Type III

mL of unlabeled GDF10. Subsequent chem- mRNA levels 0.005 ical cross-linking of [125I]TGF-β1 to its recep- 100 kDa tors, SDS/PAGE, and autoradiography indi- 0.000 cate that GDF10 competes with TGF-β1for ALK4 ALK5 ALK7 β 75 kDa binding to TβRI (55 kDa), TβRII (70 kDa), and T RII betaglycan (type III receptor). Competition Wild type Tgfbr1-/- TβRI with 100 ng/mL of cold TGF-β served as a 50 kDa GDF10, min 0 10 30 0 10 30 positive control. (Right) An identical gel was Autoradiograph Commassie pSmad3 staining stained with Coomassie blue to determine Smad3 protein loading. (F) GDF10 activation of Smad3 is TβRI-dependent. Expression of type I TGF-β receptor mRNAs in WT and Tgfbr1−/− MEFs was quantified by real-time qPCR and confirmed the expression of ALK4 and ALK7 and loss of TβRI/ALK5 in Tgfbr1−/− MEFs. (Right) Treatment with 50 ng/mL GDF10 increased pSmad3 levels in WT, but not Tgfbr1−/−, − − MEFs. (G) GDF10 activation of Smad3 is TβRII-dependent. pSmad3 was quantified by Western blot analysis in WT and Tgfbr2 / MEFs. Treatment with 50 ng/ − − mL of GDF10 increased pSmad3 in WT, but not Tgfbr2 / , MEFs.

control cells, and Sca-1 abrogated this effect (Fig. 4A). This in- result suggests that the level of activation of TGF-β signals hibition also was reflected in the activation of Smad3-mediated may depend on the relative abundance of GDF10 and Sca-1, as transcription in response to GDF10 (Fig. 4B). Because Sca-1 is well as on a possible competition between GDF10 and Sca1 for a GPI-anchored cell surface protein, we evaluated its colocaliza- receptor binding. tion with TβRI and TβRII in 34T cells by confocal microscopy (Fig. 4C). Sca-1 colocalized with TβRI and TβRII at the plasma Discussion membrane (Fig. 4C, Insets). We next examined whether Sca-1 Sca-1 is generally considered to be a stem cell marker of normal interacted with TβRI and TβRII at the cell surface. 34T cells were tissues, where Sca-1–positive cells have been shown to have a re- labeled with membrane-impermeable biotin and cross-linked, and generative capacity (3, 20–26), despite the fact that its function is Sca-1 was immunoprecipitated. Immunoblotting revealed that poorly described. In this study, we have defined a role for Sca-1 in Sca-1 was associated with biotinylated TβRI, but not with TβRII tumor progression based on three key findings. First, Sca-1 was (Fig. 4D). necessary for maintaining the tumorigenicity of mammary tumor The association of Sca-1 with TβRI at the cell surface raised the cells. Its tumor-promoting role is associated with inhibited ex- possibility that Sca-1 could prevent ligand-induced TβRI and pression of GDF10, a minimally characterized TGF-β family li- TβRII complex formation, which would explain the inhibition of gand that acts as a tumor suppressor in our tumor isograft assays. ligand-induced Smad3 activation (Fig. 4 A and B). This implies Second, GDF10 acted through TβRI and TβRII and conferred that D8 cells, with their down-regulated Sca-1 expression and high Smad3 activation similarly to, but weaker than, TGF-β1. GDF10 GDF10 expression, would have a higher level of receptor complex was previously linked to the BMP-like action of osteoblast dif- than 34T cells, and indeed this was the case (Fig. 4E). In addition, ferentiation (27, 28), but the specific receptor complex associated ectopic overexpression of Sca-1 in D8 cells disrupted the TβRI– with GDF10 had not been identified. The ability of GDF10 to act TβRII receptor complex in parallel with reduced Smad3 activation as a tumor suppressor thus resembles the well-documented role of (Fig. 4F). Finally, adding recombinant GDF10 or TGF-β1 im- TGF-β signaling as a tumor-suppressor pathway through TβRI and paired the association between Sca-1 and TβRI (Fig. 4G), sug- TβRII and Smads early in tumor development (29–31). Third, Sca- gesting that this is a dynamic interaction regulated by ligand. This 1 interacted with TβRI at the cell surface to attenuate ligand

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1103441108 Upadhyay et al. Downloaded by guest on September 26, 2021 Fig. 4. Sca-1 interacts with cell surface TGF-β receptors 25 Lysate IP: Sca-1 ABDβ ) to inhibit Smad3 activation. (A and B) NMuMG cells were GDF10 TGF- 1 3 * Control 20 Sca-1 Ctrl Sca-1 Ctrl Sca-1

transfected with either a Sca-1 or control plasmid. IgG Sca-1 IgG Sca-1 15 min 0 10 30 0 10 30 10 30 10 30 Treatment of cells with either 50 ng/mL of GDF10 or Biotinylated TβRI β pSmad3 10 WB: Strept 5 ng/mL of TGF- 1 resulted in increased pSmad3 (A) and (x10 β reporter transcription from a Smad3-responsive (TGF-β) reporter Smad2/3 5 TGF- Sca-1 0 (B) in control cells, but not in Sca-1-expressing cells. (C) GDF10 - + - + Sca-1 colocalizes with TβRI and TβRII in 34T cells. Fluo- β WB: TβRI T RI, 53 kDa rescence was visualized by confocal microscopy using C TβRI Sca-1 Merge either a TβRI or a TβRII mAb and an Alexa Fluor 488- conjugated secondary antibody or an Sca-1 mAb and an β Alexa Fluor 594-conjugated secondary antibody. The T RII, 70 kDa WB: TβRII merged images indicate that Sca-1 colocalizes with TβRI (Upper) and TβRII (Lower). Arrowheads indicate the cell membrane regions magniﬁed in the insets. (Scale bar: 10 WB: Sca-1 μm.) (D) Cell surface TβRI, but not TβRII, associates with Sca-1, 14 kDa Sca-1. Cell surface proteins in 34T cells were labeled with a membrane-impermeable biotin conjugate, and Sca-1– TβRII Sca-1 Merge interacting proteins were isolated by immunoprecipita-

tion with an Sca-1 antibody or IgG isotype control. Lysate IP: TβRI Lysate IP: TβRI Lysate Immunoprecipitated proteins were analyzed by Western EFSca-1 - + - + IgG - + D8 34T D8 IgG 34T blot using either a streptavidin-conjugated antibody for WB: pSmad3 β β biotinylated proteins or TβRI, TβRII, and Sca-1 antibodies. WB: T RII T RII WB: TβRII TβRII Sca-1 associated with a biotinylated band of the same WB: Smad2/3 β β β mobility as T RI, but not with T RII. (E) Silencing of Sca-1 WB: T RI TβRI WB: Sca-1 expression facilitates TβRI and TβRII receptor complex WB: TβRI β formation. TβRI was immunoprecipitated from lysates of T RI 34T and D8 cells, and associated TβRII was detected by Western blot analysis. D8 cells exhibited greater associ- GH β β ation of T RI with T RII compared with 34T cells. (F) Sca- Lysates IP: TβRI 1 reduces TβRI and TβRII receptor complex formation CELL BIOLOGY GDF10, min 0 5 10 - - 0 5 10 - - and Smad3 activation. D8 cells were transfected with TGF-β1, min 0 - - 5 10 0 - - 5 10 IgG

a Sca-1 or control plasmid and cell lysates were immu- WB: Sca-1 noprecipitated with a TβRI antibody, and TβRI and TβRII were detected by Western blot analysis. (Left) Over- WB: TβRI expression of Sca-1 inhibited the association of TβRI with TβRII. (Right) It also reduced pSmad3 levels. (G) Associ- WB: pSmad3 ation kinetics of Sca-1 with TβRI. 34T cells were treated WB: Smad2/3 with either 50 ng/mL of GDF10 or 5 ng/mL of TGF-β1, and TβRI was immunoprecipitated. Sca-1, TβRI, pSmad3, and Smad2/3 were detected by Western blot analysis. Both GDF10 and TGFβ1 increased pSmad3, although phosphorylation was greater in the presence of TGFβ1. Sca-1 was found to be associated with TβRI in the ab- sence of ligand, which was reduced after treatment with GDF10 or TGF-β1, whereas dissociation occurred more rapidly in the presence of TGF-β1. (H) Schematic representation of the regulation of TGF-β signaling by Sca-1. Sca-1 is depicted as interacting with TβRI to interfere with TβRI–TβRII complex formation and the subsequent activation of Smad3. When Sca-1 expression is reduced, GDF10 expression is increased, leading to stabilization of the receptor complex and Smad3 activation.

binding to the TβRI/TβRII receptor complex and ligand-activated The finding that Sca-1 promotes tumor growth through sup- Smad3 signaling, thus providing a complementary mechanism to pression of GDF10-dependent TGF-β signaling may be partic- inhibit tumor suppression by TGF-β or GDF10 signaling. ularly relevant to breast cancer. TGF-β signaling through TβRI These findings support a model in which Sca-1 counters the in- and TβRII, and then through Smad2 and Smad3 as intracellular hibitory role of TGF-β signaling in mammary gland and tumor cells effectors (16, 41, 42), is known to exert an inhibitory effect on (Fig. 4H). Tumor growth suppression by Sca-1 occurs through an mammary gland development and tumorigenesis. Increased ex- autocrine mechanism, as is apparent from the marked suppression pression of activated TGF-β1 in mammary epithelial cells of of colony formation by conditioned medium from tumor cells with MMTV-TGF-β1 transgenic mice results in impaired ductal reduced Sca-1 expression. Furthermore, the expression of Sca-1 elongation and involution, and disappearance of stem cells in the early in the tumorigenic process suggests a primary role in tumor- terminal buds (43). These mice are resistant to carcinogenesis initiating cells, as demonstrated by the marked reduction of tumor and exhibit markedly suppressed tumor formation when crossed growth in Sca-1 knockdown cells. The role of Sca-1 in tumorigenesis with MMTV-TGFα mice (44). Conversely, conditional loss of may be especially relevant to our understanding of the link between β Sca-1 expression and tumor formation (7, 9, 32) and metastatic TGF- 1 expression in MMTV-PymT mice accelerates tumor – formation and metastasis (45). However, it should be noted that behavior (6, 10, 33), as well as to the radiation resistance of Sca-1 β– positive mammary epithelial progenitor cells (34, 35). In relation to after inactivation of TGF- induced growth inhibitory signaling, β our findings, expression of dominant-negative TβRII was found to increased TGF- production and autocrine signaling help pro- increase Sca-1 expression in mammary epithelial cells (36), al- mote cancer progression through their effects on the microen- though no mechanism for this effect was presented. Although, Sca- vironment and their ability to promote epithelial plasticity, which 1 is murine-specific, the Ly6 gene family has been associated with results in invasion and metastasis (29–31). In summary, we the 8q24.3 amplicon in breast cancer (37–39), and expression of the conclude that Sca-1 maintains the growth and invasive charac- homologs Ly6D and Ly6K has been linked to basal cell and meta- teristics of tumor cells in part by suppressing the expression of static breast cancer (37–39), as well as to head and neck cancer (40). GDF10 and in part by attenuating the accessibility of the TGF-β

Upadhyay et al. PNAS Early Edition | 5of6 Downloaded by guest on September 26, 2021 receptor complex for ligand binding, both of which result in in- gether with the GFP shRNA or Sca-1 shRNA construct. At 24 h after hibition of TGF-β signaling through GDF10. transfection, the medium was replaced and virus was collected. 34T cells were infected with lentivirus for 24 h in the presence of 4 mg/mL of polybrene, the Materials and Methods medium was changed after 24 h, and selection carried out with 2 μg/mL of More detailed information is presented in SI Materials and Methods. puromycin. Cell lines were maintained thereafter in media containing 2 μg/mL of puromycin. 34T cells were transfected with pcDNA3.1-GDF10 and selected Reagents and Plasmids. GDF10 was obtained from R&D Systems, TGF-β1 was in 1 mg/mL of G418. D8 cells were transfected with a GDF10 shRNA plasmid or 125 obtained from Sigma-Aldrich, and [ I]TGF-β1 was obtained from Perkin- a control shRNA plasmid, obtained from SA Biosciences, and cells were se- β Elmer. The Smad3-responsive TGF- (46) and Smad1/5-responsive BMP (47) lected with 1 mg/mL of G418, as well as 2 μg/mL of puromycin. reporter plasmids were provided by Dr. Peter ten Dijke, Leiden University Medical Center, Leiden, The Netherlands. ACKNOWLEDGMENTS. This work was supported by National Institutes of Health Grant R01 CA111482 and Contract N01 CN43309 (to R.I.G.) and Grant shRNAs and Cell Lines. Five Sca-1 shRNAs cloned into pLKO.1 were obtained RO1 CA136690 (to R.D.). This investigation was conducted using the Animal from Open Biosystems and screened for activity in Sca-1–expressing 293T cells; Research, Flow Cytometry, Macromolecular Analysis, and Microscopy and GFP shRNA was used as a control. shRNA D8 containing the sense sequence Imaging Shared Resources supported by National Center for Research GAA-CAA-TCT-TTG-CTT-ACC-CAT produced the greatest reduction in Sca-1 Facilities Research Facilities Improvement Grant C06 RR14567, National protein level (Fig. S2) and was used for further studies. Lentivirus was pro- Cancer Institute Cancer Center Support Grant 1P30-CA-51008, and National duced in 293T cells by cotransfection of the pMD2.g and VSVG vectors to- Institutes of Health Shared Instrumentation Grant 1 S10 RR019291-01A2.

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