A Role for Endoglin As a Suppressor of Malignancy During Mouse Skin Carcinogenesis

Research Article

A Role for Endoglin as a Suppressor of Malignancy during Mouse Skin Carcinogenesis

Eduardo Pe´rez-Go´mez,1 Marıá Villa-Morales,2 Javier Santos,2 Jose´Fernańdez-Piqueras,2 Carlos Gamallo,3 Javier Dotor,4 Carmelo Bernabeú,5 and Miguel Quintanilla1

1Instituto de Investigaciones Biome´dicasAlberto Sols, Consejo Superior de Investigaciones Cientı´ficas, 2Laboratorio de Gene´ticaMolecular Humana, Departamento de Biologı´a, and 3Hospital Universitario de la Princesa, Universidad Auto´noma de Madrid; 4DIGNA Biotech; and 5Centro de Investigaciones Biolo´gicas, CSIC, and Center for Biomedical Research on Rare Diseases, Madrid, Spain

Abstract cies (8, 9). Current evidence suggests that endoglin acts as a mole- h Endoglin is a membrane glycoprotein that acts as a coreceptor cular switch in endothelial cells by promoting TGF- signaling for transforming growth factor-B. We and others have pre- through ALK1, which phosphorylates Smad1/5 and leads to the viously suggested a function of endoglin as a tumor sup- stimulation of cell migration and proliferation. In the presence of low levels of membrane-bound endoglin, TGF-h signals through pressor in epithelial cancer. Here, we study the expression of h endoglin during chemical mouse skin carcinogenesis. We find a distinct T RI, ALK5, which activates Smad2/3 and inhibits cell that shedding of membrane endoglin, allowing the secretion proliferation and migration (10, 11). of a soluble endoglin form, is a late event associated with prog- In addition to the predominant long (L) isoform of endoglin, we ression from squamous to spindle cell carcinomas. Knock- have previously reported the expression of an alternative shorter down of endoglin in transformed keratinocytes activates the (S) splice variant, called S-endoglin, in human and mouse tissues Smad2/3 signaling pathway resulting in cell growth arrest, (12, 13). These two endoglin isoforms share the extracellular and delayed tumor latencies, and a squamous to spindle pheno- transmembrane domains and only differ from each other in their typic conversion. Forced expression of the long endoglin iso- cytoplasmic tails. Studies with a transgenic mouse model that form in spindle carcinoma cells blocks transforming growth targets S-endoglin expression to the endothelium suggest an anta- factor-B1 stimulation of Smad2/3 signaling and prevents gonistic antiangiogenic role for this variant with respect to the tumor formation. In contrast, expression of the short endoglin L-endoglin form (13). isoform has no effect on spindle cell growth in vitro or in vivo. Endoglin is highly expressed in endothelial cells of the tumor Our results show that endoglin behaves as a suppressor of vasculature and at much lower levels in tumor cells. Besides the malignancy during the late stages of carcinogenesis. There- proangiogenic role of endoglin in endothelial cells (7–10), there is fore, disruption of membrane endoglin emerges as a crucial also evidence supporting its involvement in malignant progres- event for progression to spindle cell carcinomas. [Cancer Res sion by its direct function on the tumor cells themselves. Thus, loss 2007;67(21):10268–77] of endoglin expression in cultured human prostate cancer cells enhances cell migration and invasiveness (14). Also, we have À reported that endoglin-heterozygous (Eng+/ ) mice exhibit accel- Introduction erated malignant progression during chemical mouse skin Endoglin (CD105) is a homodimeric cell membrane glycoprotein carcinogenesis in vivo (13, 15). h h that is considered as a transforming growth factor- (TGF- ) The two-stage chemical protocol (16) involves treatment of mice auxiliary receptor (1). Several data support an important role for with a single dose of a carcinogen; i.e., 7,12-dimethylbenz(a)- endoglin in vascular development and physiology. Endoglin-null anthracene (DMBA), followed by repeated applications of the À/À (Eng ) mouse embryos die at midgestation due to cardiac and tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). vascular defects (2–4). In addition, the endoglin gene is a target for This treatment gives rise to benign papillomas, some of which the disorder hereditary hemorrhagic telangiectasia type I (HHT-1), progress to malignant squamous cell carcinomas (SCC). SCCs can a vascular dysplasia characterized by mucocutaneous telangiecta- be classified into three different groups according to the degree sias and arteriovenous malformations (5). A second target gene for of squamous differentiation (17): well differentiated (grade 1), h hereditary hemorrhagic telangiectasia (HHT-2) is the TGF- type I moderately differentiated (grade 2), and poorly differentiated h receptor (T RI) activin receptor–like kinase I (ALK1; ref. 6), which (grades 3/4). Some pathologists include spindle cell carcinomas h points to a close relationship between endoglin and TGF- sig- (SpCC), a highly anaplastic tumor predominantly formed by elong- naling in endothelial cells (7). Endoglin is overexpressed in endo- ated or spindle-shaped cells, into the group of poorly differentiated thelial cells during tumor neoangiogenesis, and several studies have SCCs. The physiologic inducer that pushes SCC towards SpCC defined this glycoprotein as a powerful marker to quantify intra- seems to be TGF-h1 (16). Several data support a causal relation- tumoral microvessel density in solid and hematopoietic malignan- ship between TGF-h1 and the SCC-SpCC transition. First, transformed keratinocytes cultured in the presence of TGF-h1 undergo a reversible epithelial-mesenchymal transition (EMT) associated Note: Supplementary data for this article are available at Cancer Research Online with the SCC-SpCC transition in vivo (18–20). Second, targeted (http://cancerres.aacrjournals.org/). h Requests for reprints: Miguel Quintanilla, Instituto de Investigaciones Biome´dicas expression of TGF- 1 in the epidermis of transgenic mice has a Alberto Sols, Consejo Superior de Investigaciones Cientı´ficas-Universidad Auto´noma dual effect during two-stage carcinogenesis, by inhibiting the early de Madrid, Arturo Duperier 4, 28029 Madrid, Spain. Phone: 34-9158-54412; Fax: 34- appearance of benign papillomas, but increasing malignant 9158-54401; E-mail: [email protected]. I2007 American Association for Cancer Research. progression to SpCC (21). These and other studies support the doi:10.1158/0008-5472.CAN-07-1348 notion of a double role for TGF-h1 in carcinogenesis, by acting as

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Endoglin and Malignant Progression a suppressor at early stages of tumorigenesis, but also as a sti- Western blot and flow cytometry analysis. Western blot analyses in À mulator of malignancy at later stages (16, 22). The fact that Eng+/ lysates obtained from tumors and cell lines were done as reported (15). mice exhibit an identical double phenotype to that of mice over- The primary antibodies used in this study are described in Supplementary Â 5 expressing TGF-h1 during carcinogenesis led us to postulate a role Table S3. For flow cytometry analysis, cells (5 10 ) were incubated with the anti-human endoglin monoclonal antibody (mAb) P4A4 (30) for for endoglin in skin tumorigenesis by attenuating TGF-h1 signaling 30 min at 4jC. Alexa Fluor 488–labeled anti-mouse IgG (Molecular Probes) in keratinocytes (15). More importantly, this result suggested that was used as secondary antibody. Fluorescence was estimated with a BD endoglin could act as a suppressor of malignancy during multi- FACScan using logarithmic amplifiers. stage carcinogenesis. These hypotheses have been addressed here Cell proliferation, migration, and invasion assays. For cell growth by analyzing the expression of endoglin mRNA and protein dur- assays, cells left at confluence for 24 h were plated in triplicate onto glass Â 4 ing mouse skin carcinogenesis in vivo and in vitro. In addition, coverslips at a density of 5 10 cells/60 mm dish with or without TGF-h1. we have investigated the functional effects of endoglin knockdown Seventy-two hours later, cells were trypsinized and counted. Incorporation in transformed keratinocytes and endoglin overexpression in of 5-bromo-2¶-deoxyuridine (BrdUrd) into DNA was also determined by spindle carcinoma cells. using the Cell Proliferation ELISA BrdUrd kit (Roche Diagnostic). In vitro wound healing and Matrigel invasion assays were done as described (31). Tumorigenicity assays. For tumorigenicity assays, f106 viable cells Materials and Methods were i.d. injected into the two flanks of 10-week-old nonobese diabetic- Cell culture conditions and treatments. Epidermal cell lines severe combined immunodeficiency mice, as detailed in Supplementary (Supplementary Table S1) were grown as described elsewhere (15). CarED Table S4. The size of tumors was calculated from caliper measurements of was derived by explanting a poorly differentiated carcinoma induced in an two orthogonal diameters at different times. Tumors were excised in two À Eng+/ mouse by DMBA/TPA (15). CarED cells are spindle-shaped in vitro regions and immediately frozen in liquid nitrogen (for RT-PCR and Western and produce SpCC with latency periods of f4 weeks when injected into blot analyses) or fixed in 10% formaldehyde (for histologic and immunodeficient mice. immunohistochemical characterization). RNA and protein was extracted For TGF-h1 treatments, recombinant human TGF-h1 (PreproTech EC) from the frozen tumors using Tripure reagent (Roche Diagnostic). was added to the culture medium at 10 ng/mL. The ThRI kinase inhibitor SB431542 (Sigma-Aldrich) was added to the cells at 10 Amol/L, and the antagonists of TGF-h binding P144 and P17 (DIGNA Biotech) were used at Results 200 Ag/mL. Membrane-bound endoglin is shed during the squamous to Conditioned medium was obtained by growing cells in serum-free in vivo conditions (f106 cells/mL) for 48 h. Proteins were then concentrated by SpCC transition . The levels of endoglin transcripts present cold acetone at À20jC, and the presence of soluble endoglin determined by in the normal epidermis and chemically induced skin tumors were Western blotting. determined by quantitative RT-PCR (Fig. 1A). Two primer sets were Chemical carcinogenesis, tumor characterization, and immunohis- used to amplify sequences either common to L- and S-endoglin tochemistry. All animal experiments were approved and done according to mRNAs or specific for S-endoglin mRNA. The levels of S-endoglin institutional guidelines for animal care. Induction of tumors by initiation transcripts in all samples were negligible with respect to the values with DMBA and promotion with TPA for 15 weeks has been described (15). obtained using primers that simultaneously amplify both endoglin A total of 20 Swiss albino mice were used, from which 2 mice were left isoforms (data not shown), confirming the absence of S-endoglin in untreated to obtain normal epidermis. Groups of two to four animals the epidermis (13). Endoglin mRNA expression was reduced f5- to bearing tumors were sacrificed at 15, 30, 38, and 43 weeks post-DMBA 10-fold in papillomas relative to normal epidermis. These levels initiation. Tumors were excised, divided into two regions, and immediately frozen in liquid nitrogen or fixed in 10% formaldehyde and embedded in remained relatively low in early papillomas (15 weeks post- paraffin. Tumors were histologically typed by H&E staining of paraffin initiation), late papillomas (38 weeks), and well/moderately sections, and by reverse transcription-PCR (RT-PCR) analysis of the differentiated SCCs, but increased during progression to poorly following differentiation/progression markers: K1 and K10, which are lost differentiated carcinomas to almost equalize with those of the in SCCs (23); the extracellular matrix protein SPARC, which is induced normal epidermis (Fig. 1A). during progression from papillomas to SCC (24); and the E-cadherin Endoglin expression was also studied by Western blotting using transcriptional repressor Snail, which accumulates in poorly differentiated the specific mAb MJ7/18, which recognizes the NH2-terminal SCCs (25). region of the mouse endoglin ectodomain (32). A specific band of Stable transfections and reporter assays. For short interfering RNA f130 kDa corresponding to the membrane monomer (mEng) (siRNA)–mediated knockdown of endoglin, double-stranded oligonucleo- was detected in the normal epidermis (Supplementary Fig. S1). tides encoding siRNA that silence the mouse endoglin gene (10) were inserted into the pSUPER-GFP vector. The pSUPER-GFP Thermotoga The expression profile of mEng during carcinogenesis was rather maritima vector containing an endoglin-unrelated sequence (Qiagen) was sinuous. The amount of mEng decreased in early papillomas used as a control. Transfections were carried out using LipofectAMINE plus paralleling the mRNA data, but rose to normal values in late (Invitrogen) according to the manufacturer’s instructions. Individual clones papillomas to diminish again in well and moderately differentiated were isolated using a FACS Vantage cell sorter (Becton Dickinson). Stable carcinomas. Interestingly, mEng was undetectable in poorly transfections with human L- and S-endoglin cDNAs subcloned into the differentiated carcinomas (Fig. 1A), despite the relatively high pcDNA3 vector were carried out as previously described (13). levels of endoglin transcripts in these tumors. We also observed the h Reporter assays with TGF- –responsive promoters were done as de- presence of endoglin polypeptides of f50 to 65 kDa in the tumors scribed elsewhere (11), using p(CAGA)12-luc (26) and pARE-Fast-luc (27, 28) (Supplementary Fig. S1). These smaller endoglin forms correspond constructs, in the presence or absence of TGF-h1, as indicated. The pRL-tk to a soluble form of endoglin (sEng; ref. 33). sEng was absent from Renilla normalizing luciferase vector (Promega) served as an internal control to correct for transfection efficiency and for normalization. normal epidermis and the levels of sEng found in papillomas and Quantitative and semiquantitative RT-PCR analysis. Semiquantita- early carcinomas were relatively low. However, the production of tive and quantitative RT-PCR analyses were carried out as described sEng increased significantly in poorly differentiated carcinomas, (13, 29). The oligonucleotides and PCR conditions used in these studies are and this enhancement occurred concomitantly with the disap- described in Supplementary Table S2. pearance of mEng (Fig. 1A). Immunostaining in paraffin-embedded www.aacrjournals.org 10269 Cancer Res 2007; 67: (21). November 1, 2007

Figure 1. Endoglin expression during mouse skin carcinogenesis. A, transcriptional expression of endoglin in normal epidermis and tumors induced by DMBA/ TPA (top). Normalized endoglin transcripts levels were measured by quantitative RT-PCR relative to glyceraldehyde-3-phosphate dehydrogenase transcript levels. Columns, means from three independent experiments; bars, SD. Quantification of membrane endoglin (mEng) and soluble endoglin (sEng) protein expression (middle and bottom). The intensities of the 130-kDa (mEng) and 50 to 65 kDa (sEng) bands of the blot in Supplementary Fig. S1 (at the top of the corresponding graphs) were quantified by densitometric analysis and normalized to h-actin, used as a control for protein loading. Ep, normal epidermis; Pap, papilloma; SCC, squamous cell carcinoma. All SCCs were obtained at 43 wk post-initiation, except the tumor on lane 16 which was excised at 38 wk. B, immunohistochemical detection of endoglin in skin tumors. A membrane-bound endoglin form (mEng) was detected in a 15-week-old papilloma, which corresponds to that of lane 3 in the blots in A. A circulating soluble form of endoglin (sEng) was detected in the stroma (s) and inside of blood vessels (*) in a SCC 3/4 corresponding to that of lane 22 in the blots in A. t, tumor mass; e, hyperplastic epidermis adjacent to the tumor. C, endoglin expression in mouse epidermal cell lines. The normalized endoglin transcript levels (arbitrary value = 1 for MCA3D), as measured by quantitative RT-PCR (top). Representative Western blot of mEng in whole cell lysates. No sEng form was found in the blot (bottom). a-Tubulin was used as a control for protein loading. D, sEng is produced in spindle carcinoma cells. Western blot analysis of endoglin in cell lysates and conditioned media of MCA3D and CarC cells. sections revealed the presence of endoglin at the plasma mem- panel of cell lines corresponding to different stages of carcinogen- brane of papillomas, but we were unable to detect membrane- esis; i.e., papilloma, SCC and SpCC (Supplementary Table S1). bound endoglin in poorly differentiated carcinomas. Instead, Overall, endoglin mRNA expression in these epidermal cell lines in these tumors, intense endoglin staining was seen dispersed was highly reduced with respect to the skin in vivo, with levels throughout the stroma and filling the lumen of blood vessels f1% of those found in normal epidermis. The level of endoglin (Fig. 1B), indicating the presence of a cleaved endoglin ectodomain transcripts varied among the different cell lines regardless of its that contained the epitope recognized by the MJ7/18 mAb. Alto- origin or malignant potential (Fig. 1C). The SpCC cell line CarED gether, these results suggest that mEng is shed from the surface had the lowest levels of endoglin transcripts, as it was derived from À of skin tumor cells to produce a circulating sEng form, and that a DMBA/TPA-induced skin carcinoma in an Eng+/ mouse. shedding occurs, i.e., during the progression from SCC to SpCC. Nevertheless, the rest of the SpCC cell lines (CarC, CarB, and Spindle carcinoma cells produce soluble endoglin in vitro. MSC11A5) expressed substantial amounts of endoglin mRNA In order to confirm whether endoglin shedding was associated with comparable to those expressed in papilloma and SCC cell lines. the spindle phenotype, we analyzed the expression of endoglin in a Western blot analysis revealed the presence of the 130-kDa

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Endoglin and Malignant Progression mEng monomer in premalignant keratinocytes (MCA3D, PB, and CarC cells because of the lack of antibodies reacting with the endo- MSC11P6) and SCC cell lines (MSC11B9, PDV, HaCa4, Pam212, glin cytoplasmic tail. and CarC-R), but we were unable to detect mEng in the four siRNA-mediated knockdown of endoglin in transformed spindle cell lines analyzed (Fig. 1C), as occurs in vivo. Among these keratinocytes promotes EMT. In order to examine whether cell lines, each pair formed by MSC11B9/MSC11A5 and CarC-R/ loss of endoglin expression has any effect on the phenotype of CarC represents a homogeneous cell system for malignant squamous carcinoma cells, we chose the PDV cell line that exhibits progression. Both MSC11B9 and MSC11A5 cell lines were derived an epithelial phenotype in vitro and produces well to moderately from the same carcinoma, B9 from the squamous component and differentiated SCCs upon injection in mice (Supplementary A5 from the anaplastic spindle region (34). Similarly, CarC-R Table S1). PDV cells were stably transfected with a vector encoding represents a minor, less aggressive epithelial cell subpopulation siRNA sequences that specifically suppress endoglin expression isolated from the spindle carcinoma cell line CarC (35). Because (10). As a control, an irrelevant siRNA was used (siCont). Two mEng was present in MSC11B9 and CarC-R but not in MSC11A5 clones (siEng2 and siEng4) that showed reduced mRNA expres- and CarC (Fig. 1C), these results suggest that ‘‘loss’’ of the 130-kDa sion (Fig. 2A) and synthesized about a half and a third of the membrane endoglin form is a specific event associated with the endogenous endoglin protein levels were obtained (Fig. 2B). In squamous to spindle transition. In addition, we were able to detect contrast to control cells, siEng cells had an elongated shape, grew sEng in the conditioned medium of CarC, but not in the dispersed, and were unable to form cohesive islands at low density conditioned medium of MCA3D keratinocytes that express mEng (Fig. 2B, bottom). Consistent with these results, siEng2 and siEng4 (Fig. 1D). These data indicate that loss of mEng in spindle cells is clones showed diminished levels of epithelial proteins, such as due to shedding. We could not assess whether truncated mEng E-cadherin, and induced the expression of mesenchymal/progres- lacking part or all the ectodomain was present at the surface of sion markers (Fig. 2B; Supplementary Fig. S2 and Supplementary

Figure 2. Endoglin down-regulation in transformed PDV keratinocytes promotes an EMT. A, expression of endoglin and E-cadherin transcripts by RT-PCR in PDV cells stably transfected with siRNA endoglin (siEng) or siRNA control (siCont). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as a control for the amount of cDNA present in each sample. B, Western blot analysis of mEng and of differentiation/progression markers in siCont and siEng transfectants. The extent of mEng protein reduction in siEng cells is indicated. a-Tubulin was used as a control for protein loading. Phase contrast micrographs of siCont and siEng cells (bottom). C, siEng are more sensitive than siCont cells to TGF-h1–induced complete EMT. Western blot analyses of E-cadherin and vimentin expression at different times of treatment with TGF-h1. D, TGF-h1 stimulates mEng shedding in siCont cells. Western blot analysis of endoglin in cell lysates (top) and conditioned media (bottom) of siCont cells at different times of treatment with TGF-h1. *, nonspecific band corresponding to an abundant secreted polypeptide unrelated to endoglin. www.aacrjournals.org 10271 Cancer Res 2007; 67: (21). November 1, 2007

Information). Down-regulation of E-cadherin protein did not the growth factor stimulates endoglin shedding prior to TGF- involve a transcriptional mechanism, as the levels of E-cadherin h1–induced EMT. mRNA expressed in siEng clones were similar to that of control siRNA-mediated knockdown of endoglin in transformed cells (Fig. 2A). keratinocytes activates Smad2/3 signaling and decreases cell PDV cells elicit a complete EMT when grown in the presence growth in vitro and in vivo. In order to examine whether of TGF-h1, but it requires the continuous stimulus of the growth endoglin modulates TGF-h signaling in transformed keratinocytes, factor for >2 weeks (18). However, siEng cells were more sen- we analyzed the effect of endoglin knockdown on TGF-h1–induced sitive to TGF-h1–induced complete EMT than control cells, as Smad phosphorylation. In the absence of TGF-h1, siCont and siEng monitored by Western blot analysis of epithelial (E-cadherin) and cell transfectants showed low amounts of phosphorylated Smad2 spindle (vimentin) markers (Fig. 2C). We also analyzed whether (P-Smad2) and P-Smad3 (Fig. 3A). However, the levels of P-Smad2 TGF-h1–mediated EMT involved down-regulation of mEng. As and P-Smad3 (normalized by the total amounts of protein) were shown in Fig. 2D, TGF-h1 promoted the progressive ‘‘disappear- slightly higher in siEng clones with respect to control cells, as seen ance’’ of mEng in siCont cells and the concomitant appearance by a longer exposure of the blot (data not shown). TGF-h1 stimu- of sEng in the conditioned medium. At 7 days of treatment lated Smad2 and Smad3 phosphorylation in both siCont and siEng with TGF-h1, loss of mEng was almost complete, indicating that cells, but P-Smad2 and P-Smad3 levels were higher in endoglin

Figure 3. Endoglin down-regulation in PDV keratinocytes leads to enhanced Smad2/3 signaling, cell growth inhibition, and a SCC-SpCC conversion. A, Western blot analysis of Smad2/3 phosphorylation (P-Smad) relative to the total expression levels at different times of treatment with TGF-h1. Representative of two independent experiments. B, TGF-h1–induced transcriptional activation of pARE-Fast and pCAGA luciferase reporter genes. Cells were transiently transfected with the abovementioned reporters and treated or not with TGF-h1 for 24 h. Transcriptional activity was measured using the luciferase reporter assay. A representative experiment using triplicate samples corrected for transfection efficiency out of three. C, cell growth assays in the absence or presence of TGF-h1. Synchronized cells were seeded in culture dishes and grown in medium plus serum in the absence or presence of TGF-h1. After 72 h, cells were trypsinized and counted. Columns, means of triplicate incubations; bars, SD. D, tumorigenic potential of siCont and siEng cells. Cells were injected into the two flanks of nonobese diabetic-severe combined immunodeficiency mice, and the sizes of the tumors induced by the cell lines were measured every 2 to 3 d. Right, average values from six tumors for each cell line. Left, the expression of E-cadherin and vimentin was analyzed by Western blot on samples isolated from individual tumors. Tumors were excised at 38 d post-injection. Detection of immunoglobulins present in the tumors served as a control for protein loading.

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Figure 4. Endoglin down-regulation in PDV keratinocytes stimulates cell migration and invasiveness. A, in vitro wound-healing assay. Wounded confluent cell cultures were cultured for 24 h with or without TGF-h1, in the absence or presence of either the ThRI kinase inhibitor SB431542 (10 Amol/L) or the TGF-h antagonist peptide P144 (200 Ag/mL). Quantification of migrated cells was done by measuring the healed area (n = 5). Data are representative of three independent experiments. B, Matrigel invasion assay. Serum-starved cells were seeded on the upper compartments of Matrigel invasion chambers. Serum (10%) and TGF-h1 (where indicated) were used as chemoattractants. SB431542 and P144 inhibitors were added to both the upper and lower compartments of the chamber at the concentrations described in A. Cells that went through the Matrigel-coated filter after 24 h were counted by submerging the underside of the filter in a 4¶,6-diamino-2-phenilindole solution (10 Ag/mL) in order to stain nuclei. Columns, means of four fields from duplicated assays; bars, SD. Represents one out of three independent experiments. C, treatment with SB431542, but not with P144, promotes a reversion to the epithelial phenotype. Phase contrast micrographs of siCont and siEng4 cells incubated for 24 h in the presence (or absence) of either SB431542 or P144 (top and middle). The expression of E-cadherin in siEng4 cells was detected by immunofluorescence analysis (bottom). siEng4 cells treated with SB431542 shows the typical keratinocyte E-cadherin staining at cell-cell contacts, whereas untreated cells or cells treated with P144 exhibit an aberrant E-cadherin cytoplasmic localization. knockdown cells. Consistent with these results, we found that both siRNA-mediated knockdown of endoglin in transformed basal and TGF-h1–stimulated activities of the pARE-Fast and pCAGA keratinocytes stimulates cell migration and invasiveness. reporter constructs, which are specific for Smad2 and Smad3 The phenotypic changes observed in siEng cells were associated activation, respectively, were significantly higher in siEng cells with increased basal and TGF-h1–stimulated migratory and inva- (Fig. 3B). Basal pARE-Fast and pCAGA luciferase activities were f2- sive abilities (Fig. 4A and B). To test whether the enhanced basal to 3-fold higher in siEng clones compared with siCont cells, whereas migratory potential of siEng cells involved the activation of TGF-h1 was able to stimulate pARE-Fast and pCAGA transactiva- TGF-h signaling, two distinct TGF-h antagonists, SB431542 and tion by 9- and 150-fold, respectively. These results indicate that P144, were used. SB431542 is a potent inhibitor of the kinase reduced endoglin expression leads to increased basal and TGF- activity of ThRI and blocks Smad2/3 signaling mediated by ALK5 h1–stimulated Smad2/3 signaling in transformed keratinocytes. (38). P144 is a betaglycan-derived soluble peptide that inhibits the TGF-h1 is a potent inducer of keratinocyte growth arrest (36), a binding of TGF-h1 to its receptors (39). Interestingly, treatment response that seems to be mediated by the Smad2/3 pathway with SB431542, but not with P144, reduced the basal cell migration (22, 37). In this regard, increased Smad2/3 signaling in siEng cell and invasiveness of siEng cells to the levels exhibited by siCont clones is compatible with their slow growth and increased cells. In contrast, both antagonists blocked TGF-h1–stimulated sensitivity (f3-fold) to TGF-h1–induced growth inhibition com- cell migration and invasiveness of siCont cells (Fig. 4A and B). pared with control cells (Fig. 3C). Similar results were obtained Identical results to those raised with P144 were obtained with P17, by measuring the incorporation of BrdUrd into DNA (data not another antagonist peptide of similar characteristics as P144 (data shown). Reduced growth of siEng cells was also observed in vivo not shown). These data show that activated Smad2/3 signaling in after injection of the cells into immunodeficient mice. siEng endoglin–down-regulated keratinocytes results in enhanced migra- tumors grew slower and had longer latency periods than tumors tion/invasiveness. Furthermore, activation of the Smad2/3 pathway induced by control cells (Fig. 3D; Supplementary Table S4). Never- is not triggered by an autocrine mechanism involving elevated theless, they were vimentin-positive and E-cadherin–negative secretion of TGF-h1, neither by up-regulation of ALK5 expres- (Fig. 3D) and were typed as SpCCs, whereas siCont tumors were sion (see below). Instead, down-regulation of endoglin expression well/moderately differentiated SCCs, the same phenotype as leads to the intrinsic activation of ALK5 at the cell surface, allow- tumors induced by the parental cell line (18). These results suggest ing enhanced Smad2/3 signaling and EMT. As a matter of fact, that down-regulation of endoglin expression in transformed siEng cells reverted to the epithelial phenotype by blocking ThRI keratinocytes simultaneously leads to decreased cell growth and activity with SB431542, but not by preventing the binding of a SCC-SpCC conversion in vivo. TGF-h1 with P144 (Fig. 4C). www.aacrjournals.org 10273 Cancer Res 2007; 67: (21). November 1, 2007

Figure 5. Effect of the ectopic expression of human L- and S-endoglin on the behavior of spindle CarC cells. A, the expression of exogenous L- and S-endoglin in the transfectants was analyzed by flow cytometry. Top right, percentages of cells expressing endoglin at the cell surface. B, TGF-h1–induced transcriptional activation of pARE-Fast and pCAGA luciferase reporter genes. Experimental conditions were as in the legend of Fig. 3B. C, tumorigenic potential of CarC cell transfectants. Points, means of all tumors induced by the three clones corresponding to each transfected isoform, as detailed in Supplementary Table S4; bars, SD. D, characterization of exogenous endoglin expression in tumors induced by the cell transfectants. Tumors induced by the S-Eng and neo cell transfectants were excised at 21 d post-injection, whereas those induced by L-Eng transfectants were excised at 31 d post-injection. The expression of either L-endoglin or S-endoglin was analyzed by RT-PCR (top) and Western blot (middle). Amplification of glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) was used as a control for the amount of cDNA present in each sample, whereas detection of immunoglobulins present in the tumors served as a control for protein loading. Bottom, the production of sEng in the conditioned media of cell lines (passage = 2) derived from explantation of neo and L-Eng tumors was analyzed by Western blot. *, a nonspecific band unrelated to endoglin that partially masks sEng.

Expression of endoglin in spindle carcinoma cells inhibits Although expression of L- or S-endoglin did not significantly Smad2/3 signaling and suppresses tumorigenicity. Next, we alter the fibroblastic morphology of CarC cells (Supplementary studied whether forced expression of endoglin affected the Fig. S3B), studies with specific reporter genes showed that phenotype of spindle carcinoma cells. To this aim, CarC cells were L-endoglin blocked TGF-h1 stimulation of Smad2/3 signaling transfected with cDNAs encoding the human L and S endoglin (Fig. 5B). Interestingly, S-endoglin did not only fail to inhibit tran- isoforms. Clones that stably expressed L- and S-endoglin (L-Eng sactivation of the reporter genes but seemed to enhance f2-fold and S-Eng, respectively) were obtained by cell sorting. A pool of the TGF-h1 stimulation of pCAGA luciferase activity (Fig. 5B), CarC cells transfected with the empty vector was used as a control. indicating that the presence of S-endoglin in CarC cells favors Interestingly, flow cytometry analysis using the specific mAb P4A4 TGF-h1/Smad3 signaling. CarC cells are not growth-inhibited by directed against the human endoglin ectodomain (30) showed the TGF-h1 (36), and the expression of L- or S-endoglin did not presence of cell surface L-endoglin in only 53% to 55% of L-Eng significantly affect the growth properties of CarC cells in vitro (data cells, whereas membrane S-endoglin was detected in the vast not shown). However, L-endoglin had a remarkable effect on the majority of S-Eng cells (Fig. 5A). Similar results were obtained growth of spindle CarC cells in vivo. Tumors induced by L-Eng by Western blot analysis of mEng expression (Supplementary clones had latency periods 3- to 4-fold longer than those induced Fig. S3A). Because small sEng forms were detected in the con- by S-Eng and control cells (Fig. 5C; Supplementary Table S4). This ditioned media of L-Eng but not of S-Eng transfectants (data not difference was confirmed by injecting an additional third trans- shown), the lower surface expression of L-endoglin relative to fectant clone for each isoform (L-Eng3 and S-Eng3). All neo, L-Eng, S-endoglin in the transfectants seems to be due to shedding. In and S-Eng tumors were histologically typed as SpCCs (Supplemen- addition, these data suggest that the short isoform is refractory to tary Table S4; Supplementary Fig. S3C). When the expression of shedding. Another compelling observation is derived from the endoglin was analyzed in vivo, significant levels of L-endoglin comparative analyses of the size of endoglin protein expressed in transcripts were observed in tumors induced by L-Eng trans- distinct cell types (see Supplementary Information). fectants, but we were unable to detect L-endoglin protein by

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Western blot (Fig. 5D) or immunohistochemistry (Supplementary ability to interfere with TGF-h signaling. From the two known Fig. S3C). In contrast, S-endoglin protein was easily detected in the endoglin isoforms, it is the L form which is relevant for epidermal tumors induced by S-Eng cells. Furthermore, no L-endoglin protein carcinogenesis; S-endoglin is barely expressed during carcinogen- was detected in the cell lysates of explanted L-Eng tumors, whereas esis in vivo and in vitro, and it does not seem to have any influence smaller sEng forms were found in the conditioned media of these on the growth or invasiveness of tumor cells. cells (Fig. 5D, bottom). In the case of L-Eng tumors, these results An important finding in the present study is that endoglin indicate that only those CarC cells that have lost surface expression shedding from the membrane, which disrupts mEng function and of L-endoglin allow their growth in vivo, suggesting that those cells releases soluble sEng into the stroma, is an event associated with which actively shed mEng are selected for tumor formation. progression to the highly invasive spindle tumor phenotype in vivo. ALK5 expression is reduced in endoglin–down-regulated The proteolytic cleavage of the ectodomain is a phenomenon that cells. As the expression of ThRI and ThRII have been reported to occurs in many transmembrane proteins. For example, shedding of À À À change in Eng / and Eng+/ endothelial cells (10, 40, 41), we betaglycan, another TGF-h coreceptor, disrupts the enhancer analyzed whether similar changes occur in our PDV and CarC cell activity of TGF-h binding exhibited by the membrane-bound form transfectants. ALK5 mRNA expression was highly reduced in and produces a soluble protein that inhibits TGF-h binding to its endoglin-down-regulated PDV keratinocytes, whereas the expres- surface receptors (42). Similarly, sEng has been involved in the sion of ThRII was not affected (Fig. 6A). Both ALK5 and ThRII pathogenesis of the pregnancy-specific hypertensive syndrome levels were down-regulated in CarC compared with PDV cells, and preeclampsia by preventing binding of TGF-h1 to its receptors and neither L-endoglin nor S-endoglin modified the expression of both impairing downstream signaling activity (33, 43). sEng is also found receptors (Fig. 6B). Reduced levels of ALK5 and ThRII might in the sera and/or plasma of patients with different neoplasias (9). explain the weaker response of CarC cells to pARE-Fast and pCAGA More importantly, elevated levels of serum sEng (in other words, reporter genes compared with PDV keratinocytes (2.5- and 7-fold enhanced endoglin shedding) correlates with metastasis in patients stimulation in CarC versus 9- and 150-fold in PDV, respectively). with colorectal, breast, and other solid malignancies (44, 45). These No expression of the endothelial-specific ThRI ALK1 was found in observations are in line with our finding that endoglin shedding is the cell lines. a late event in carcinogenesis associated with the development of poorly differentiated carcinomas, and suggest that the tumor Discussion suppressor function of mEng, as determined in this work, might also be relevant for human cancer. The enzyme(s) (sheddases) We show in this work that the TGF-h coreceptor endoglin plays a involved in the release of the endoglin ectodomain are presently crucial role in skin carcinogenesis by modulating tumor cell growth unknown. Nevertheless, a clue for the identification of this and invasiveness. This role of endoglin most likely derives from its sheddase(s) comes from the fact that TGF-h1 stimulates endoglin shedding in cultured keratinocytes, and therefore, the enzyme(s) involved might be regulated by TGF-h1. Although our in vivo and in vitro data point to neoplastic cells as a key contributor to sEng, the role of stromal cells in sEng production cannot be ruled out. Further studies are necessary to determine the relative contribu- tion of the distinct tumor cell components to sEng and whether sEng itself has any influence on tumor development. To study the functional implications of down-regulating mEng function in tumor cells, we used siRNA technology. Our data indicate that endoglin attenuates TGF-h1/Smad2/3 signaling in keratinocytes. Knockdown of endoglin in transformed keratinocytes stimulated basal and TGF-h1–mediated ALK5/Smad2/3 signaling activity. Interestingly, basal ALK5 activation was innate and did not involve enhanced binding of TGF-h or increased expression of the receptors. ALK5 activation promoted EMT and the in vivo development of a spindle phenotype associated with enhanced cell migration/invasiveness. These results are in accordance with the fact that progression to poorly differentiated À carcinomas was vastly accelerated during carcinogenesis in Eng+/ mice (13, 15), and strongly indicate that mEng behaves as a suppressor of malignancy at late stages of carcinogenesis. Con- sequently, disruption of mEng by shedding emerges as a crucial event for TGF-h1–mediated SCC-SpCC transition in vivo. Interest- ingly, a recent report (46) shows that endoglin also regulates EMT in the developing heart during embryogenesis. Down-regulation of mEng in transformed keratinocytes leads

Figure 6. Expression of ALK5, ALK1, and ThRII transcripts in siRNA endoglin to cell growth arrest in vitro and in vivo, likely because of increased PDV cell transfectants (A) and human L- and S-endoglin CarC cells transfectants ALK5/Smad2/3 signaling. Keratinocytes with reduced mEng levels (B) by RT-PCR. Amplification of glyceraldehyde-3-phosphate dehydrogenase down-regulate ALK5 expression probably as an adaptation (GAPDH) was used as a control for the amount of cDNA present in each sample. ÀRTlane, the results of amplification in the absence of cDNA. RNA isolated from mechanism in order to proliferate, as reported in endothelial cells murine lung was used as a positive control for ALK1 expression. lacking endoglin (10). The fact that growth inhibition occurs www.aacrjournals.org 10275 Cancer Res 2007; 67: (21). November 1, 2007

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Cancer Research concomitantly with enhanced cell migration and invasiveness keratinocyte cell growth, the significance of the reduction of mEng indicates that the tumor suppressor and oncogenic functions of expression observed in early papillomas could be to counteract the TGF-h overlap. Therefore, loss of the TGF-h growth-inhibitory chronic proliferative response of keratinocytes to TPA (16). Thus, in À response seems to be a prerequisite for SCC to progress to SpCC. Eng+/ mice, or in mice overexpressing TGF-h1 in the epidermis, Most cells derived from SCCs remain responsive to the TGF-h chemical carcinogenesis produces a much reduced papilloma load growth-inhibitory response, and resistance to the inhibitory effects (15, 21). of the growth factor seems to occur late during carcinogenesis, Interestingly, forced expression of L-endoglin was shown to sup- which is associated with the spindle phenotype (36, 47). Several press the growth of CarC spindle cells in vivo but not in vitro.In mechanisms might account for the lack of an antiproliferative contrast, S-endoglin was innocuous for CarC cell growth. The mole- response in tumor cells harboring no mutations in the TGF-h cular basis for this effect remains to be determined, but seems to receptors or Smads (22, 37). Thus, unresponsive spindle CarC cells reside in the cytoplasmic tail of the molecule, as it is the sole domain have deleted the Ink4b locus encoding the cyclin-dependent kinase which is different between L- and S-endoglin. On the other hand, inhibitor p15 (48), a key mediator of the TGF-h antiproliferative S-endoglin specifically stimulated TGF-h1/Smad3 signaling, an response (37). CarC cells, however, respond to the TGF-h1 stimu- opposite action with respect to L-endoglin, reinforcing our sug- lation of Smad2/3 signaling and, therefore, have an intact TGF-h/ gestion that L- and S-endoglin isoforms exert antagonistic roles (13). Smad pathway. Nevertheless, the magnitude of stimulation of h the Smad2/3 pathway by TGF- 1 in CarC is lower than in PDV Acknowledgments cells, likely because CarC cells have down-regulated both ALK5 and ThRII expression. We postulate that loss-of-function of mEng Received 4/16/2007; revised 7/18/2007; accepted 8/30/2007. Grant support: Spanish Ministry of Education and Science grants SAF2004-04902 must exert a strong selective pressure during tumor evolution to (M. Quintanilla) and SAF2004-01390 (C. Bernabe´u), and a fellowship from the prime cells that have missed or inactivated components of the Autonomous Community of Madrid (E. Pe´rez-Go´mez). h The costs of publication of this article were defrayed in part by the payment of page TGF- antiproliferative response. These selected cells will be prone charges. This article must therefore be hereby marked advertisement in accordance to TGF-h1–mediated EMT giving rise to SpCCs. This could be with 18 U.S.C. Section 1734 solely to indicate this fact. the reason why loss of the TGF-h1 antiproliferative response We are in debt to Dr. Peter ten Dijke for kindly providing the plasmid for endoglin knockdown as well as TGF-h reporters. We also thank Drs. Amelia Nieto and David cosegregates with the highly invasive spindle phenotype. As down- Sarrio´for their gifts of plasmids and antibodies, respectively, and Eva G. Santos for regulation of endoglin expression leads to the inhibition of skilful technical assistance.

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