Oncogene (2011) 30, 1046–1058 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Endoglin expression in breast tumor cells suppresses invasion and metastasis and correlates with improved clinical outcome LA Henry1, DA Johnson1, D Sarrio´1, S Lee1, PR Quinlan2, T Crook1, AM Thompson2, JS Reis-Filho1 and CM Isacke1 1Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, London, UK and 2Department of Surgery and Molecular Oncology, Ninewells Hospital and Medical School, Dundee, UK Tumor growth factor-b (TGF-b) signaling in cancer has been and metastatic events in late stage carcinomas (Roberts implicated in growth suppression of early lesions and and Wakefield, 2003; Massague, 2008). TGF-b ligands act enhancing tumor cell invasion and metastasis. However, by binding to their cognate transmembrane serine/ the cellular mechanisms that determine this signaling output threonine kinase receptors (Shi and Massague, 2003; Feng in individual tumors are still largely unknown. In endothelial and Derynck, 2005). This results in the activation of the cells, TGF-b signaling is modulated by the TGF-b co- canonical TGF-b pathway mediated by the phosphoryla- receptor endoglin (CD105). Here we demonstrate that tion of Smad proteins and their subsequent translocation endoglin is expressed in a subset of invasive breast cancers to the nucleus where they promote the transcriptional and cell lines and is subject to epigenetic silencing by gene regulation of TGF-b response genes. However, TGF-b can methylation. Endoglin downregulation in non-tumorigenic also signal through a range of other pathways (Moustakas MCF10A breast cells leads to the formation of abnormal and Heldin, 2005; Zhang, 2009) and these alternate acini in 3D culture, but does not promote cell migration or pathways are particularly important in the pro-invasive transformation. In contrast, in the presence of activated responses to TGF-b in cancer (Bhowmick et al., 2001; ErbB2, endoglin downregulation in MCF10A cells leads to Ozdamar et al., 2005). enhanced invasion into a 3D matrix. Consistent with these Endoglin (CD105) is a type I integral transmembrane data, ectopic expression of endoglin in MDA-MB-231 cells glycoprotein that has predominantly been investigated blocks TGF-b-enhanced cell motility and invasion and in the vasculature where it is upregulated during reduces lung colonization in an in vivo metastasis model. angiogenesis (Bernabeu et al., 2007, 2009). In endothe- Unlike endothelial cells, endoglin does not modulate Smad- lial cells, endoglin associates at the cell surface with the mediated TGF-b signaling in breast cells but attenuates the type I and type II TGF-b receptors and has a key role in cytoskeletal remodeling to impair cell migration and modulating downstream signaling to the Smad transdu- invasion. Importantly, in a large cohort of invasive breast cer proteins (Guerrero-Esteo et al., 2002; Lebrin et al., cancers, lack of endoglin expression in the tumor cell 2004). The importance of endoglin acting as a TGF-b compartment correlates with ENG gene methylation and co-receptor in these events is evidenced by the observa- poor clinical outcome. tion that mutations in either ENG or the type I receptor Oncogene (2011) 30, 1046–1058; doi:10.1038/onc.2010.488; ALK1 give rise to the vascular disorder hereditary published online 1 November 2010 hemorrhagic telangiectasia (McAllister et al., 1994; Johnson et al., 1996). From an initial demethylation Keywords: endoglin; CD105; breast cancer; TGF-b; screen, we identified ENG as a candidate gene for MCF10A; ErbB2 epigenetic regulation in breast cancer cell lines. Together with previous reports that endoglin can inhibit the migration of other epithelial tumors (Craft et al., 2007; Perez-Gomez et al., 2007), we hypothesized that Introduction expression of endoglin might function to suppress breast cancer progression. To address this we investigated the The tumor growth factor-b (TGF-b) signaling pathway functional role of endoglin in both in vitro and in vivo has an important role in all stages of breast cancer models, its impact on the TGF-b signaling pathway and progression. It is well established that TGF-b has a dual the prognostic impact and regulation of endoglin function in acting both as a tumor suppressor in the early expression in a large cohort of invasive breast cancers. stage of malignant progression and to promote invasive Results Correspondence: Professor CM Isacke, Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, 237 Fulham Road, Downregulation of endoglin expression enhances breast London SW3 6JB, UK. E-mail: [email protected] cancer cell invasion Received 30 March 2010; revised 31 August 2010; accepted 3 September A panel of cell lines was subject to immunoblotting and 2010; published online 1 November 2010 quantitative real-time PCR (qPCR). Endoglin mRNA Endoglin suppresses breast cancer invasion LA Henry et al 1047 100 Endoglin MCF10A 75 (Long exposure) Endoglin 100 75 (Short exposure) shEng1 shEng5 shEng2 shCont Parental 50 Tubulin 100 Endoglin 75 50 37 ERK1/2 Relative mRNA expression p=0.0145 p=0.0141 p=0.1927 Parental shCont shEng2 % of abnormal acini shEng1 shEng5 shCont shEng1 shEng2 shEng5 Parental Figure 1 Endoglin downregulation enhances abnormal MCF10A acini formation in 3D culture. (a) Endoglin expression analyzed in a series of breast cell lines by immunoblotting (upper panels) or qPCR (lower panel). Data shown are the mean±s.e.m. of RQ relative expression of two independent experiments. (b) Immunoblotting of MCF10A cells stably infected with different shRNA lentiviral constructs against endoglin (shEng) or control (shCont). Note, the shEng2 cell line showed negligible endoglin knockdown and therefore was used as an independent shRNA control. (c) MCF10A cell lines stably expressing control shRNA (shCont, shEng2) or endoglin knockdown (shEng1, shEng5) shRNA constructs were grown in 3D culture and fixed on day 20. Cells were permeabilized and stained for collagen IV and Alexa488 anti-rabbit Ig (green), the Golgi protein b-COP and Alexa555 anti-rat Ig (red) and DAPI (blue). Left panel, representative confocal images. Scale bar, 25 mm. Right panel, mean % of abnormal acini morphology for three independent experiments±s.e.m. and protein expression was readily detected in the non- endoglin lentiviral small hairpin RNAs (shRNAs) tumorigenic breast cell line MCF10A, in a subset of the (shEng1–5) targeting different regions of the ENG gene cancer cell lines (MDA-MB-468, SKBR3, T47D) and in or with control shRNA (shCont) (Figure 1b). Substan- the recently re-classified melanoma line MDA-MB-435 tial knockdown was observed with shEng1 and shEng5 (Figure 1a). This expression was 2.5–5-fold lower than but not with shEng2. The shEng2 and shCont cell lines found in the cultured endothelial cells (Supplementary expressed similar levels of endoglin as the parental line Figure 1). No endoglin expression was detected in and hence were used as two independent controls. In MCF7, Cal51, MDA-MB-231, MDA-MB-453, HCC1954 this 3D culture model, shCont and shEng2 cells were or BT474 cells. Reverse transcription–PCR analysis indistinguishable from the parental MCF10A cells and revealed that in the endoglin-positive cell lines the formed single acini that contained a hollow lumen predominant expression was of the long-endoglin isoform surrounded by a polarized layer of epithelial cells with with little or no expression of the short-endoglin isoform their Golgi orientated inwards (Figure 1c). In contrast, detected (data not shown). the endoglin-knockdown lines (shEng1 and shEng5) To explore a potential role for endoglin as a breast produced abnormally shaped acini in which the outer cancer suppressor, we initiated this study with the non- layer of cells was not fully polarized and the lumena tumorigenic MCF10A cell line. MCF10A cells when were incompletely cleared. Nonetheless, these shEng1 grown on a thick layer of Matrigel form epithelial acini and shEng5 acini, still growth arrested, did not invade (3D culture model), and this system has been used into the Matrigel and were no larger than control extensively to identify pathways that can perturb the structures indicating that the loss of endoglin expression normal breast architecture (Debnath and Brugge, 2005). alone does not result in the transformation of the non- MCF10A cells were stably infected with five individual tumorigenic MCF10A cells. Oncogene Endoglin suppresses breast cancer invasion LA Henry et al 1048 Cell line Expression MCF10A.ErbB2 parental 100% MCF10A.ErbB2 shCont 91.1% MCF10A.ErbB2 shEng1 33.2% MCF10A.ErbB2 shEng2 71.0% MCF10A.ErbB2 shEng5 4.7% p=0.0126 p=0.0428 Parental shEng1 p=0.0403 p=0.0087 p=0.7998 shCont shEng5 % of acini with invasive protrusions -+++ - +++ -+++ +++ -+++- AP1510 --+--+- --+----+- -+- - TGF-β1 shEng2 ---+--+ ---+---+---- + SB431542 Parental shContshEng2 shEng1 shEng5 Figure 2 Endoglin downregulation cooperates with ErbB2 activation to promote invasion in 3D culture. (a) MCF10A.ErbB2 cells were infected with shCont or shEng lentiviral constructs. Stable cell lines were subject to FACS analysis with an isotype control or endoglin antibody. The mean level of fluorescence signal (FITC-A) relative to the parental cells is shown. Note, the shEng2 cell line has negligible endoglin knockdown and therefore was used as an independent shRNA control. (b) MCF10A.ErbB2 cell lines were grown in 3D culture for 5 days±TGF-b1±SB431542±AP1510 dimerizer added from day 4. Acini were stained for laminin V and Alexa488 anti-mouse Ig (green), Alexa546-phalloidin (red), and DAPI (blue). Left panel, representative confocal images of cultures with AP1510 dimerizer added from day 4 in the absence of TGF-b1 and SB431542. Arrowheads indicate invasion protrusions and laminin V degradation. Scale bar, 25 mm. Right panel, mean % of acini with invasive protrusions for three independent experiments±s.e.m. We next tested whether endoglin might function to AP1510-treated cultures produced acini with signifi- modulate the effects of activated oncogenes.