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TGFβ-mediated suppression of CD248 in non-cancer cells via canonical Smad-dependent signaling pathways… Suresh Babu, Sahana; Valdez, Yanet; Xu, Andrea; O’Byrne, Alice M; Calvo, Fernando; Lei, Victor; Conway, Edward M Feb 20, 2014

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RESEARCH ARTICLE Open AccessTGFβ-mediated suppression of CD248 innon-cancer cells via canonical Smad-dependentsignaling pathways is uncoupled in cancer cellsSahana Suresh Babu1, Yanet Valdez1, Andrea Xu1, Alice M O’Byrne1, Fernando Calvo2,3, Victor Lei1and Edward M Conway1*AbstractBackground: CD248 is a cell surface glycoprotein, highly expressed by stromal cells and fibroblasts of tumors andinflammatory lesions, but virtually undetectable in healthy adult tissues. CD248 promotes tumorigenesis, while lackof CD248 in mice confers resistance to tumor growth. Mechanisms by which CD248 is downregulated are poorlyunderstood, hindering the development of anti-cancer therapies.Methods: We sought to characterize the molecular mechanisms by which CD248 is downregulated by surveyingits expression in different cells in response to cytokines and growth factors.Results: Only transforming growth factor (TGFβ) suppressed CD248 protein and mRNA levels in cultured fibroblasts andvascular smooth muscle cells in a concentration- and time-dependent manner. TGFβ transcriptionally downregulatedCD248 by signaling through canonical Smad2/3-dependent pathways, but not via mitogen activated protein kinases p38or ERK1/2. Notably, cancer associated fibroblasts (CAF) and cancer cells were resistant to TGFβ mediated suppressionof CD248.Conclusions: The findings indicate that decoupling of CD248 regulation by TGFβ may contribute to its tumor-promotingproperties, and underline the importance of exploring the TGFβ-CD248 signaling pathway as a potential therapeutictarget for early prevention of cancer and proliferative disorders.BackgroundCD248, also referred to as endosialin and tumor endothe-lial marker (TEM-1) [1] (reviewed in [2]), is a member ofa family of type I transmembrane glycoproteins containingC-type lectin-like domains, that includes thrombomodulin[3] and CD93 [4]. Although the mechanisms are not fullyelucidated, these molecules all modulate innate immunity,cell proliferation and vascular homeostasis and are poten-tial therapeutic targets for several diseases, including can-cer, inflammatory disorders and thrombosis.CD248 is expressed by cells of mesenchymal origin, in-cluding murine embryonic fibroblasts (MEF), vascularsmooth muscle cells, pericytes, myofibroblasts, stromal cellsand osteoblasts [5-12]. During embryonic development,CD248 is prominently and widely expressed in the fetus(reviewed in [2]). However, after birth, CD248 protein levelsare dramatically downregulated [7,13-15], resulting in onlyminimal expression in the healthy adult, except in theendometrium, ovary, renal glomerulus and osteoblasts[11,16-18].While largely absent in normal tissues, CD248 is mark-edly upregulated in almost all cancers. Highest expressionis found in neuroblastomas and in subsets of carcinomas,such as breast and colon cancers, and in addition, in glio-blastomas and mesenchymal tumors, such as fibrosarco-mas and synovial sarcomas [8,14,15,17,19,20], where it ismostly detected in perivascular and tumor stromal cells,but also in the tumor cells themselves [21,22]. CD248 isalso expressed in placenta and during wound healing andin wounds such as ulcers. It is also prominently expressedin synovial fibroblasts during inflammatory arthritis [10].In some tumors and in chronic kidney disease, CD248* Correspondence: ed.conway@ubc.ca1Centre for Blood Research, Department of Medicine, University of BritishColumbia, 4306-2350 Health Sciences Mall, V6T 1Z3, BC Vancouver, CanadaFull list of author information is available at the end of the article© 2014 Suresh Babu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons PublicDomain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in thisarticle, unless otherwise stated.Suresh Babu et al. BMC Cancer 2014, 14:113http://www.biomedcentral.com/1471-2407/14/113expression directly correlates with worse disease and/or apoor prognosis [9,23,24]. The contributory role of CD248to these pathologies was confirmed in gene inactivationstudies. Mice lacking CD248 are generally healthy, exceptfor an increase in bone mass [11,25] and incomplete post-natal thymus development [26]. However, in several models,they are protected against tumor growth, tumor invasive-ness and metastasis [25,27] and they are less sensitive toanti-collagen antibody induced arthritis [10].While the mechanisms by which CD248 promotestumorigenesis and inflammation are not clearly defined,the preceding observations have stimulated interest in ex-ploring CD248 as a therapeutic target, primarily by usinganti-CD248 antibodies directed against its ectodomain[19,20,28,29]. Likely due to limited knowledge of CD248regulatory pathways, other approaches to interfere with orsuppress CD248 have not been reported. CD248 is upreg-ulated in vitro by high cell density, serum starvation, bythe oncogene v-mos [5] and by hypoxia [30]. We previ-ously showed that fibroblast expression of CD248 is sup-pressed by contact with endothelial cells [27]. Otherwise,factors which down-regulate CD248 have not heretoforebeen reported, yet such insights might reveal novel sitesfor therapeutic intervention.In this study, we evaluated the effects of several cyto-kines on the expression of CD248. We show that TGFβspecifically and dramatically downregulates CD248 ex-pression in normal cells of mesenchymal origin and thatthis is mediated via canonical Smad-dependent intracellu-lar signaling pathways. Notably, cancer cells and cancerassociated fibroblasts are resistant to TGFβ mediated sup-pression of CD248. The findings suggest that CD248 notonly promotes tumorigenesis, but may be a marker of thetransition of TGFβ from a tumor suppressor to a tumorpromoter. Delineating the pathways that couple TGFβ andCD248 may uncover novel therapeutic strategies.MethodsReagentsRabbit anti-human CD248 antibodies (Cat no #18160-1AP) were from ProteinTech (Chicago, USA); goat anti-human actin antibodies (#sc-1616) from Santa Cruz(USA); rabbit anti-SMAD1,5-Phospho (Cat no #9516),rabbit anti-Smad2-Phospho (#3101), rabbit anti-ERK1/2-phospho (#9101S), rabbit anti-p38-phospho (#9211),rabbit anti-SMAD2/3 (#5678) and rabbit anti-SMAD3(#9513) were from Cell Signaling (USA). Murine anti-rabbit α-smooth muscle actin monoclonal antibodies(#A5228) were from Sigma-Aldrich (Canada). Secondaryantibodies included goat anti-rabbit IRDye® 800 (LIC-926-32211). Goat anti-rabbit IRDye® 680 (LIC-926-68071) ordonkey anti-goat IRDye® 680 antibodies (LIC-926-68024) and anti-rabbit Alexa green-488 were from Licor(Nebraska, USA).Basic fibroblast growth factor (bFGF), recombinant hu-man transforming growth factor β-1 (TGFβ) (240-B/CF),recombinant human bone morphogenic protein (BMP-2)(355-BM-010/CF), recombinant human/mouse/Rat ActivinA, CF (338-AC-010/CF), recombinant rat platelet derivedgrowth factor-BB (PDGF) (250-BB-050), recombinant hu-man vascular endothelial growth factor (VEGF), andrecombinant mouse interleukin-6 (IL-6) (406-ML/CF),recombinant mouse tumor necrosis factor-α (TNF-α)(410-MT/CF) and recombinant mouse interferon-γ(IFN-γ) (485-MI/CF) were purchased from R&D Systems(Minneapolis, USA). Phorbol 12-Myristate 13-Acetate(PMA) (P1585) and α-amanitin were from Sigma-Aldrich(Oakville, Canada). The inhibitors SB431542 (for ALK5),SB202190 (for p38) and U0126 (for ERK1/2) were fromTocris Biosciences, Canada.MiceTransgenic mice lacking CD248 (CD248KO/KO) were previ-ously generated and genotyped as described [10]. Micewere maintained on a C57Bl6 genetic background and cor-responding sibling-derived wild-type mice (CD248WT/WT)were used as controls.Cell cultureMurine embryonic fibroblasts (MEF) were isolated fromCD248WT/WT or CD248KO/KO mice as previously described[10]. Cells were cultured in DMEM (Invitrogen, Canada)with 10% fetal calf serum (FCS) and 1% Penicillin/Strepto-mycin (Invitrogen, Karlsruhe, Germany) and used at pas-sages 2-5. Upon reaching confluence, cells were incubatedfor 14 hrs in low serum media (1% FCS) and then treated asindicated in the Results with TGFβ (0.1-12 ng/ml), BMP-2(50-100 ng/ml), PDGF (50 ng/ml), VEGF (20 ng/ml), bFGF(10 ng/ml), IL-6 10 ng/ml), PMA (60 ng/ml), SB43152(1 μM), and/or α-amanitin (20 μg/ml), for different time pe-riods as noted. Using previously reported methods [31,32],vascular smooth muscle cells (SMC) were isolated from theaortae of CD248WT/WT or CD248KO/KO pups, cultured inSMC growth media (Promocell, Heidelberg, Germany) with15% FCS and 1% Penicillin/Streptomycin (Invitrogen)and used at passages 2-5. Wehi-231 and A20 (mouse B-lymphoma) cell lines (gift of Dr. Linda Matsuuchi, Univer-sity of British Columbia) were cultured in RPMI media with10% fetal calf serum (FCS), 1% Penicillin/Streptomycin and0.1% mercaptoethanol. Normal fibroblasts (NF) derivedfrom normal mouse mammary glands, and cancer associ-ated fibroblasts (CAF) from mammary carcinoma in micecontaining the MMTV-PyMT transgene [33] were providedby Dr. Erik Saha (Cancer Research London UK ResearchInstitute, London, UK), and cultured in DMEM with 10%FCS, 1% Penicillin/Streptomycin and 1% insulin-transferrin-selenium.Suresh Babu et al. BMC Cancer 2014, 14:113 Page 2 of 11http://www.biomedcentral.com/1471-2407/14/113Protein electrophoresis and western blottingCells were scraped from culture dishes, suspended in PBS,pelleted by centrifugation and lysed with 50 μl RIPA buffer(30 mM Tris–HCl, 15 mM NaCl, 1% Igepal, 0.5% deoxy-cholate, 2 mM EDTA, 0.1% SDS). Centrifugation-clearedlysates were quantified for protein content. Equal quan-tities of cell lysates (25 μg) were separated by SDS-PAGEunder reducing or non-reducing conditions as noted,using 8% and 12% low-bisacrylamide gels (acrylamide tobis-acrylamide = 118:1). In pilot studies, these gels pro-vided highest resolution of the bands of interest [34]. Pro-teins were transferred to a nitrocellulose membrane andafter incubating with blocking buffer (1:1 PBS:Odyssey buf-fer) (Licor, Nebraska, U.S.A.), they were probed with rabbitanti-CD248 antibodies 140 μg/ml, goat anti-actin anti-bodies, rabbit anti-Smad1-Phospho, anti-Smad2-Phospho,anti-Smad2-Total or anti-Smad3 antibodies in blocking buf-fer overnight. After washing and incubation of the filterwith the appropriate secondary antibodies (100 ng/mlIRDye® 800 goat anti-rabbit or IRDye® Donkey anti-goat–Licor, Nebraska, USA) in blocking buffer for 1 hr at roomtemperature, detection was accomplished using a LicorOdyssey® imaging system (Licor, Nebraska, USA) and inten-sity of bands of interest were quantified relative to actinusing Licor software (Licor, Nebraska, U.S). All studies wereperformed a minimum of 3 times, and representative West-ern blots are shown.Immunofluorescence analysisPreconfluent cells were grown on cover slips and fixed atroom temperature with acetone (100%) for 2 minutes,followed by a 30 minute incubation with blocking buffer(1% BSA in PBS). Cells were then incubated with anti-CD248 rabbit antibodies 40 μg/ml, for 1 hr followed by ex-tensive washes and incubation with Alexa green 488 anti-rabbit antibody (5 mg/ml) for 1 hr. The cells were washedand fixed with antifade containing DAPI (Invitrogen,Canada) for subsequent imaging with a confocal micro-scopic (Nikon C2 model, Nikon, Canada).Determination of stability of CD248 mRNAα-Amanitin, an inhibitor of RNA-polymerase II, was usedto quantify the half-life of CD248 mRNA using previouslyreported methods [35]. Briefly, 90% confluent MEF wereincubated with DMEM with 1% fetal calf serum (FCS)overnight, after which the media was refreshed, and subse-quently stimulated with α-Amanitin 20 μg/ml ± TGFβ forthe indicated time periods. RNA was isolated for gene ex-pression analysis.Gene expression analysisRNA was isolated from the MEF and reverse transcribedto cDNA/mRNA according to the manufacturer’s in-structions (Qiagen RNeasy kit and QuantiTech reversetranscription kit, Hilden, Germany). Expression of CD248mRNA was analyzed by RT-PCR and quantified withSYBR green using real time PCR (Applied Biosystems®Real-Time PCR Instrument, Canada). CD248 mRNA levelswere reported relative to the expression of the housekeep-ing gene, Glyceraldehyde 3-Phosphate dehydrogenase(GAPDH). The following amplification primers were used:CD248 forward (5′-GGGCCCCTACCACTCCTCAGT-3′);CD248 reverse (5′-AGGTGGGTGGACAGGGCTCAG-3′);GAPDH forward (5′-GACCACAGTCCATGCCATCACTGC-3′); GAPDH reverse (5′-ATGACCTTGCCCACAGCCTTGG-3′).Animal careExperimental animal procedures were approved by theInstitutional Animal Care Committee of the Universityof British Columbia.StatisticsExperiments were performed in triplicate and data wereanalyzed using Bonferroni post-test to compare replicates(GraphPad Prism software Inc, California, USA). Errorbars on figures represent standard errors of the mean(SEM). P < 0.05 was considered statistically significant.ResultsScreen for cytokines that modulate expression of CD248In view of the established links between CD248 and cellproliferation, migration and invasion, we screened anumber of growth factors, cytokines and PMA for ef-fects on the expression of CD248 by MEF. These factorsand the chosen concentrations were selected based onthe fact that all reportedly induce MEF to undergo in-flammatory, migratory and/or proliferative changes. Wepreviously determined that these cells express CD248 atreadily detectable levels, as assessed by Western blot,where it is often seen as a monomer (~150 kDa) and adimer (~300 kDa). An incubation time of 48 hrs waschosen based on our previous findings that CD248-dependent release and activation of matrix metallopro-teinase (MMP9) induced by TFGβ was observed overthat period [10]. As seen in Figure 1A, bFGF, VEGF,PDGF, PMA, IL-6, TNF-α, and IFN-γ had no effects onCD248 expression. However, TGFβ suppressed expres-sion of CD248 in MEF to almost undetectable levels(Figure 1A). The same pattern of response was evidentin the murine fibroblast cell line 10 T1/2 (Figure 1B),and in mouse primary aortic smooth muscle cells (SMC)(Figure 1C), suggesting that CD248 specifically respondsto TGFβ and that the response is active in diverse celllines.Suresh Babu et al. BMC Cancer 2014, 14:113 Page 3 of 11http://www.biomedcentral.com/1471-2407/14/113TGFβ suppresses expression of CD248 by MEFTGFβ exerts a range of cellular effects by binding to andactivating its cognate serine/threonine kinase receptors,TGFβ type I (TGFβRI, ALK-5) and type II (TGFβRII),which in turn mediate intracellular signaling events viacanonical Smad-dependent and Smad-independent signal-ing pathways (e.g. p38 mitogen-activated protein kinase(MAPK) pathway) (for reviews [36-38]). The canonicalSmad-dependent pathway results in recruitment andphosphorylation of Smad2 and Smad3 which complexwith Smad4 to enter the nucleus and form a transcrip-tional complex that modulates target gene expression in acontext-dependent manner. Diversity in the response toTGFβ signaling is achieved by Smad2/3-independent,“non-canonical” signaling pathways, which may include,among others, activation of combinations of mitogen-activated protein kinases ERK1/2 and p38, PI3K/Akt,cyclo-oxygenase, Ras, RhoA, Abl and Src (for reviews[36-38]). We characterized the pathways by which TGFβsuppresses CD248. MEF were exposed to a range ofconcentrations of TGFβ (0.1 to 12 ng/ml) for a periodof 48 hrs. Western blots of cell lysates showed thatTGFβ downregulated the expression of CD248 in aconcentration-dependent manner. As expected, TGFβalso induced phosphorylation of Smad2 and Smad3 in aconcentration-dependent manner (Figure 2A,B). Con-focal microscopy was used to visualize the effects ofTGFβ on expression of CD248 by MEF (Figure 2C). At48 hrs without TGFβ, CD248 was readily detected on thesurface of CD248WT/WT MEF, but was entirely absent inTGFβ-treated cells as well as in CD248KO/KO MEF.We next evaluated the temporal response of CD248 to afixed concentration of TGFβ (3 ng/ml) (Figure 3A,B) andfound that CD248 expression was suppressed in a time-dependent manner to <50% by 6 hrs of exposure to TGFβ.Once again, TGFβ induced phosphorylation of Smad2.Notably, as seen in experiments using CD248KO/KO MEF(lacking CD248) (Figure 3C), CD248 was not required forTGFβ-mediated phosphorylation of Smad2, indicating thatCD248 is not a co-receptor for TGFβ signaling.TGFβ suppresses CD248 mRNA accumulationWe evaluated the mechanism by which TGFβ suppressesCD248. CD248 mRNA levels in MEF were quantified byqRT-PCR at different time intervals following exposureof the cells to 3 ng/ml TGFβ. TGFβ suppressed CD248mRNA levels in a time-dependent manner and by 75 mi-nutes, mRNA accumulation had diminished to ~50%(Figure 4) and was ~20% by 2 hrs.Using the RNA polymerase II inhibitor, α-amanitin(20 μg/ml), we measured the stability of CD248 mRNA inMEF and assessed whether it is altered by TGFβ. As seenin Figure 4, the time-dependent reduction in CD248mRNA with α-amanitin alone was almost identical to thepattern seen with TGFβ alone, i.e., the half-life was deter-mined to be approximately 75 minutes. The addition ofFigure 1 Expression of CD248 by mesenchymal cells in response to cytokines and growth factors. Murine embryonic fibroblasts (MEF)(A), 10 T1/2 cells (B) and murine aortic smooth muscle cells (SMC) (C) were incubated for 48 hrs with FGF (10 ng/ml), VEGF (20 ng/ml), PDGF(20 ng/ml), PMA (60 ng/ml), TGFβ (3 ng/ml), IL-6 (10 ng/ml), TNF-α (10 ng/ml), or IFN-γ (10 ng/ml). Cells were lysed and separated by SDS-PAGEunder non-reducing conditions for Western immunoblotting to detect CD248 and phosphorylated Smad2. Equal loading was confirmed withactin control. Only TGFβ suppressed expression of CD248, while inducing phosphorylation of Smad2. Results are representative of 3 independentexperiments. Molecular weight markers in kDa are shown on the left.Suresh Babu et al. BMC Cancer 2014, 14:113 Page 4 of 11http://www.biomedcentral.com/1471-2407/14/113TGFβ to α-amanitin did not alter the half-life. The find-ings suggest that TGFβ acts primarily at the level ofCD248 transcription and does not alter the stability ofCD248 mRNA.Suppression of CD248 by TGFβ is mediated byALK-5 signalingIn MEF, TGFβ reportedly signals exclusively through com-plexes involving ALK5 [39]. SB431542 is a selective inhibi-tor of TGFβ superfamily type I activin receptor-like kinase(ALK) receptors, ALK4, ALK5 and ALK7, which does notaffect components of the ERK, JNK, or p38 MAP kinasepathways [40]. We tested whether ALK5 is required forTGFβ-mediated suppression of CD248. MEF were incu-bated with the inhibitor (1 μM) for 1 hr prior to theaddition of 3 ng/ml TGFβ. Expression of CD248 at 48 hrswas assessed by Western blot, immunofluorescence ana-lysis and qRT-PCR (Figure 5A-C). When added alone,neither the inhibitor SB431542 nor its vehicle DMSO, hadany effect on CD248 expression. As before, TGFβ dramat-ically suppressed CD248, while simultaneously inducingphosphorylation of Smad2 (Figure 5A). This effect ofTGFβ was entirely abrogated by preincubation of the cellswith SB431542. Thus, addition of TGFβ down-regulatesCD248 via activation of ALK-5.TGFβ-mediated suppression of CD248 is independent ofERK1/2 and p38 signalingWe also tested whether suppression of CD248 expres-sion by TGFβ is mediated via one or more non-canonicalSmad2/3-independent pathways. Using U0126, a specificinhibitor of ERK1/2 phosphorylation [41], we showed thatTGFβ does not rely on signaling via ERK1/2 to suppressCD248 (Figure 6A). In a similar manner, using the p38inhibitor, SB202190 [42], we also demonstrated thatphosphorylation of p38 is not required for TGFβ todownregulate expression of CD248 (Figure 6B). Thus,in MEF, TGFβ suppresses CD248 expression via signal-ing pathways that do not require activation of these twoSmad2/3-independent pathways.Regulation of CD248 by Bone morphogenic protein 2(BMP2) and ActivinThe TGFβ family of cytokines comprises over 35 mem-bers, including the prototypic TGFβ isoforms (TGFβ1,β2, β3), bone morphogenic proteins (BMPs), growth anddifferentiation factors, activins and nodal. These regulateFigure 2 Expression of CD248 in response to increasing concentrations of TGFβ. (A) MEF were incubated for 48 hrs with increasingconcentrations of TGFβ. Expression of CD248 (seen as monomers (~160 kDa) and dimers) and phosphorylation of Smad2, were detected byWestern blot. (B) CD248 expression relative to actin expression was quantified by densitometry (n = 3 experiments) and results were normalizedto the no-treatment condition. (C) CD248 expression by MEF (wild-type, WT; or lacking CD248, KO) was detected with specific anti-CD248antibodies after exposure to carrier (Control) or TGFβ for 48 hrs. TGFβ suppresses CD248 in a concentration-dependent manner, with simultaneousincrease in phosphorylated Smad2 and ERK1/2. Scale bar = 50 μm.Suresh Babu et al. BMC Cancer 2014, 14:113 Page 5 of 11http://www.biomedcentral.com/1471-2407/14/113cell survival, proliferation, differentiation, adhesion, mi-gration and death in a cell type-and context-dependentmanner. To further assess the specificity of action ofTGFβ on CD248 expression, we tested whether BMP2and activin had similar effects. MEF were treated for 24and 48 hrs with 50 and 100 ng/ml of activin or BMP2(Figure 7A). At these concentrations of BMP2, Smad1was, as expected, phosphorylated, while Smad2 was not[43]. Notably, BMP2 had no effect on CD248 expres-sion, and thus does not participate in its regulationunder these conditions. Activin induced phosphoryl-ation of Smad2, which reportedly occurs via ALK-4/7activation [44] (Figure 7B). In contrast to TGFβ, activincaused only a slight reduction in CD248 expressionafter 48 hrs of exposure.Cancer cell lines are resistant to TGFβ suppressionof CD248Since elevated CD248 is associated with tumorigenesis, wetested whether TGFβ could suppress CD248 in tumor celllines as effectively as in the healthy non-cancerous cellsexamined above. Mouse B lymphoma cell lines, Wehi-231and A20 were incubated with TGFβ at concentrations of3 ng/ml and 12 ng/ml for 24 hrs and 48 hrs (Figure 8).Under these conditions, SMAD2 was phosphorylated, withFigure 3 Temporal response of CD248 to TGFβ. (A) MEF were incubated for 0-48 hrs with TGFβ 3 ng/ml. Expression of CD248 and phosphorylationof Smad2, were detected by Western blot. (B) CD248 expression relative to actin expression was quantified by densitometry (n = 3 experiments) and resultswere normalized to the no-treatment condition. CD248 expression decreases as Smad2 is phosphorylated. (C) CD248WT/WT (WT) or CD248KO/KO (KO) MEFwere exposed to TGFβ (0 or 3 ng/ml) for 48 hrs and lysates were Western blotted. Representative blots from 3 experiments are shown. Smad2 and ERK1/2are phosphorylated in response to TGFβ even in cells that lack CD248.Figure 4 Stability of CD248 mRNA is unaffected by TGFβ.MEF were treated with TGFβ 3 ng/ml alone, α-amanatin 20 μg/mlalone, or with a combination of TGFβ and α-amanitin as describedin Methods. CD248 mRNA levels, relative to the mRNA levels of thehousekeeping gene GAPDH, were quantified at different time intervalsby qRT-PCR. Results were normalized from 3 independent experiments,each done in triplicate. The half-life of CD248 mRNA is approximately75 minutes, which is unaltered by TGFβ.Suresh Babu et al. BMC Cancer 2014, 14:113 Page 6 of 11http://www.biomedcentral.com/1471-2407/14/113Figure 5 TGFβ-induced suppression of CD248 is mediated via canonical signaling pathways. (A, B, C) MEF were incubated for 48 hrs withTGFβ 3 ng/ml and the ALK-inhibitor SB431542 1 μM either singly or in combination. Controls included carriers for SB431542 (DMSO) or for TGFβ(0.1% BSA). (A) Western blots and (B) immunofluorescence were used to detect expression of CD248 (green). (C) CD248 mRNA levels were alsoquantified (n = 3 experiments, each in triplicate; *p < 0.05). Results indicate that TGFβ-mediated suppression of CD248 protein and mRNA requiresintegrity of canonical ALK5-Smad2 signaling pathway. Scale bar = 50 μm.Figure 6 TGFβ-mediated suppression of CD248 via ALK5 is specific. (A, B) MEF were incubated with TGFβ (3 ng/ml) for 48 hrs in thepresence or absence of the inhibitor of phosphorylated ERK1/2, U0126 10 μM (A) or phosphorylated p38, SB202190 10 μM (B). RepresentativeWestern blots from 3 independent experiments are shown and were used to assess the effect on CD248 expression. TGFβ-coupling to eitherERK1/2 or to p38 is not involved in its suppressive effects on CD248.Suresh Babu et al. BMC Cancer 2014, 14:113 Page 7 of 11http://www.biomedcentral.com/1471-2407/14/113minimal effect on Smad3 phosphorylation. In both theWehi-231 cells (Figure 8A) and the A20 cells (Figure 8B),there was no significant suppression of CD248 expressionin response to TGFβ. Indeed, in the latter, there was a slightincrease in CD248 in response to the TGFβ.We also examined the effect of TGFβ on the expressionof CD248 by normal and cancer associated fibroblasts (NFand CAF, respectively) that were derived from mousemammary tissues [33]. Protein levels of CD248 were rela-tively low in both of these cell lines, making it difficult toFigure 7 Regulation of CD248 by BMP-2 and Activin. MEF were incubated with different concentrations of BMP2 (A) or activin (B) for 24 or48 hrs. Representative Western blots from 3 independent experiments are shown and were used to assess the effect on CD248 expression.Figure 8 Regulation of CD248 in cancer cells. (A, B) Wehi-231 (A) and A20 (B) mouse lymphoma cells were incubated with differentconcentrations of TGFβ for 24 or 48 hrs and lysates were assessed by Western immunoblot. CD248 levels were minimally affected in spite ofphosphorylation of Smad2. Results are representative of 3 independent experiments. (C) Normal fibroblasts (NF) and cancer associated fibroblasts (CAF)from murine mammary tissue were exposed to TGFβ for 24 or 48 hrs and CD248 mRNA levels were quantified and normalized to levels from untreatedNF. CD248 mRNA levels in NF were significantly suppressed by TGFβ, whereas there was no effect on CD248 in CAF. *p < 0.05, n = 3.Suresh Babu et al. BMC Cancer 2014, 14:113 Page 8 of 11http://www.biomedcentral.com/1471-2407/14/113assess changes by Western blot. CD248 mRNA levels weretherefore quantified by qRT-PCR (Figure 8C). Followingexposure of the cells to 3 ng/ml or 12 ng/ml TGFβ for 24and 48 hrs, CD248 mRNA accumulation was significantlysuppressed in the NF, while in contrast, there was no ef-fect on CD248 mRNA levels in the CAF. Overall, the pre-ceding findings indicate that the expression of CD248 incancer cells is resistant to regulation by TGFβ.DiscussionSince the discovery of CD248 [45], clinical and genetic evi-dence has pointed to it as a promoter of tumor growthand inflammation (reviewed in [2]). Increased expressionof CD248 is detected in stromal cells surrounding mosttumors, and high levels often correlate with a poor prog-nosis [20,23]. Means of interfering with the tumorigeniceffects of CD248 have eluded investigators due to a lack ofknowledge surrounding the regulation of CD248. This haslimited opportunities for the design of innovative thera-peutic approaches. In this report, we show that expressionof CD248 by non-cancerous cells of mesenchymal originis specifically and dramatically downregulated at a tran-scriptional and protein level by the pleiotropic cytokine,TGFβ, and that the response is dependent on canonicalSmad2/3-dependent signaling. Notably, CD248 expressionby cancer cells and cancer associated fibroblasts is not al-tered by TGFβ. The findings suggest that a TGFβ-basedstrategy to suppress CD248 may be useful as a therapeuticintervention to prevent early stage, but not later stage,tumorigenesis.Members of the TGFβ family regulate a wide range ofcellular processes (e.g. cell proliferation, differentiation, mi-gration, apoptosis) that are highly context-dependent, i.e.,stage of development, stage of disease, cell/tissue type andlocation, microenvironmental factors, and epigenetic fac-tors. Under normal conditions, TGFβ plays a dominant roleas a tumor suppressor at early stages of tumorigenesis, inhi-biting cell proliferation and cell migration (reviewed in[46,47]). TGFβ ligands signal via TGFβRI (ALK-5) andTGFβRII. A third accessory type III receptor (TGFβRIII)lacks kinase activity, but facilitates the tumor-suppressoractivities of TGFβ. TGFβ binds to TGFβRII which trans-phosphorylates ALK-5. In canonical signaling, ALK-5 thenphosphorylates Smad2 and Smad3, inducing the formationof heteromeric complexes with Smad4, for translocationinto the nucleus, interaction with transcription factors, andregulation of promoters of several target genes [48,49]. Dis-ruption of TGFβ signaling has been associated with severalcancers and a poor prognosis [47], and mice that lack TGFβspontaneously develop tumors and inflammation [50].TGFβ signaling is not, however, restricted to Smads 2and 3, but can couple to non-canonical (Smad2/3-inde-pendent) effectors [48,51-54]. Recent data support the no-tion that canonical signaling favours tumor suppression,while non-canonical signaling tips the balance, such thatTGFβ switches to become a promoter of tumor growth, in-vasion and metastasis, overriding the tumor-suppressingactivities transmitted via Smad2/3. This dichotomous na-ture is known as the “TGFβ Paradox”, a term coined to de-scribe the conversion in function of TGFβ from tumorsuppressor to tumor promoter [55-57]. The mechanismsunderlying this switch are steadily being delineated, as regu-lation of the multiple effector molecules that are coupled toTGFβ are identified and characterized (reviewed in [47]).Our findings suggest that CD248 may be one such TGFβ-effector molecule that undergoes a context-dependentchange in coupling, and thus may be a potential therapeutictarget.Upon determining that TGFβ suppresses CD248, we firstshowed that the response is dependent on Smad 2 signal-ing. This is consistent with the almost undetectable levelsof CD248 in normal tissues, its expression presumably heldin check at least in part by TGFβ’s tumor suppressor prop-erties. The fact that TGFβ induces phosphorylation ofSmad2 in MEF that lack CD248, indicates that CD248 isnot required for Smad2 phosphorylation. Rather, in theTGFβ-signaling pathway, CD248 is positioned “down-stream” of Smad2/3 phosphorylation. We also showed thatCD248 is downregulated by TGFβ primarily at a transcrip-tional level, and without affecting the stability of its mRNA.We have not determined which regions of the CD248 pro-moter are required for TGFβ-induced suppression. How-ever, intriguingly, the murine promoter of the CD248 genecontains the sequence 5′-TTTGGCGG (position −543to −536) [5] that overlaps with a consensus E2F transcrip-tion factor binding site. This is almost identical to theunique Smad3 DNA binding site in the c-myc promoterthat is crucial for TGFβ-induced gene suppression [58]. De-tailed mapping of the promoter will provide insights intoprecisely how CD248 is regulated by TGFβ.We also examined whether TGFβ coupling to non-canonical effector molecules, ERK1/2 and p38, alters ex-pression of CD248. Neither ERK1/2 nor p38, pathwaysimplicated in TGFβ-induced metastasis, affected CD248expression. Thus, based on current data, TGFβ-inducedsuppression of CD248 occurs primarily, if not exclusively,via canonical Smad2/3 signaling.The specificity of the response of CD248 to TGFβ ex-tends beyond Smad2/3-related signaling. In a survey ofgrowth factors and cytokines, we could not identify otherfactors that similarly suppress (or conversely, increase)CD248 expression in MEF, 10 T1/2 cells or primary vascu-lar smooth muscle cells. Even BMP2 and activin, membersof the TGFβ superfamily and pleiotropic cytokines thatalso exhibit tumor promoter and suppressor activities, hadlittle effect on CD248 expression. Although our surveywas limited in range, concentration and time of exposure,the findings suggest specificity, and highlight the centralSuresh Babu et al. BMC Cancer 2014, 14:113 Page 9 of 11http://www.biomedcentral.com/1471-2407/14/113role that TGFβ likely plays in regulating expression ofCD248 in non-cancerous cells.Most notably, in two tumor cell lines and in cancer as-sociated fibroblasts, the regulation of expression of CD248was resistant to TGFβ. Indeed, in these cells, TGFβ neitherdecreased nor increased CD248, suggesting a decouplingof the regulatory link between TGFβ and CD248. Thus,with the switch from a tumor suppressor to a tumor pro-moter, TGFβ loses it ability to regulate CD248. AlthoughTGFβ does not appear to directly participate in enhancingCD248 expression during late tumorigenesis, loss of itsability to suppress CD248 may be relevant in tumor pro-gression and metastasis.ConclusionsWe have shown that the tumor suppressor properties ofTGFβ, observed in early stage cancer, are likely mediated inpart via suppression of CD248, the latter which is mediatedvia canonical Smad-dependent pathways. Upregulationof CD248 might be an early detection marker of tumorgrowth and metastasis, and may be valuable in monitoringTGFβ-based therapies. The clinical relevance of under-standing how CD248 is regulated is highlighted by ongoingPhase 1 and 2 clinical trials in which the anti-CD248 anti-body, MORAb-004, is being tested for efficacy in solid tu-mors and lymphomas (www.clinicaltrials.gov). Delineatingthe molecular mechanism(s) by which TGFβ loses its abilityto suppress CD248 will be key for the design of additionaltherapeutic interventions to prevent and/or reduce CD248-dependent tumor cell proliferation and metastasis.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsSSB helped design and perform the experiments and wrote the manuscript.YV helped design the studies and prepare the manuscript. AX, AO and VLprovided technical support. FC prepared and provided normal and cancerassociated fibroblasts. EMC supervised, directed and designed all studies andwrote the manuscript. All authors read and approved the final manuscript.AcknowledgementsWe thank Dr. Erik Sahai, Cancer Research UK London Research Institute, forinput on the manuscript and for providing cancer associated fibroblasts. FCwas supported by a Cancer Research UK grant CRUK_A5317. YV wassupported by a Michael Smith Foundation for Health Research/Crohns’ andColitis Foundation of Canada Trainee Award and is a recipient of apostdoctoral fellowship from the Canadian Institutes for Health Research(CIHR). EMC is supported by operating grants from the CIHR and the CanadaFoundations for Innovation (CFI). He holds a CSL Behring Research Chair anda Tier 1 Canada Research Chair in Endothelial Cell Biology, is an adjunctScientist with the Canadian Blood Services, and is a member of theUniversity of British Columbia Life Sciences Institute.Author details1Centre for Blood Research, Department of Medicine, University of BritishColumbia, 4306-2350 Health Sciences Mall, V6T 1Z3, BC Vancouver, Canada.2Tumour Cell Biology Laboratory, Cancer Research UK London ResearchInstitute, London, UK. 3Tumour Microenvironment Team Division of CancerBiology, The Institute of Cancer Research, London, UK.Received: 25 November 2013 Accepted: 17 February 2014Published: 20 February 2014References1. Christian S, Ahorn H, Koehler A, Eisenhaber F, Rodi HP, Garin-Chesa P, Park JE,Rettig WJ, Lenter MC: Molecular cloning and characterization of endosialin,a C-type lectin- like cell surface receptor of tumor endothelium. J Biol Chem2001, 276(10):7408–7414.2. 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Frederick JP, Liberati NT, Waddell DS, Shi Y, Wang XF: Transforming growthfactor beta-mediated transcriptional repression of c-myc is dependenton direct binding of Smad3 to a novel repressive Smad binding element.Mol Cell Biol 2004, 24(6):2546–2559.doi:10.1186/1471-2407-14-113Cite this article as: Suresh Babu et al.: TGFβ-mediated suppressionof CD248 in non-cancer cells via canonical Smad-dependent signalingpathways is uncoupled in cancer cells. BMC Cancer 2014 14:113.Suresh Babu et al. BMC Cancer 2014, 14:113 Page 11 of 11http://www.biomedcentral.com/1471-2407/14/113


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