Oncogene (2007) 26, 5290–5299 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Cortical stabilization of b- contributes to NHERF1/EBP50 tumor suppressor function

EL Kreimann1, FC Morales1, J de Orbeta-Cruz1, Y Takahashi1, H Adams2, T-J Liu1, PD McCrea3, and M-M Georgescu1,2

1Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 2Department of Molecular Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA and 3Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Anchorage-independent growth is a hallmark of tumor extracellular matrix owing to alterations in cell surface growth and results from enhanced proliferation and altered molecules such as cadherins and integrins (Thiery, cell–cell and cell-matrix interactions. By using - 2002). Cadherins constitute a major family of trans- deficient mouse embryonic fibroblasts (MEFs), we showed membrane glycoproteins that mediate cell–cell adhesion. for the first time that NHERF1/EBP50 (Na/H exchanger Adjacent cells in tissues are linked by the interaction of regulator factor 1/ezrin-radixin-moesin binding phospho- cadherin extracellular domains, but the strength of the 50), an adapter protein with membrane localization binding is supported by cytoplasmic interactions, which under physiological conditions, inhibits cell motility and is include those with and thereby the actin required to suppress anchorage-independent growth. Both cytoskeleton (Provost and Rimm, 1999). This intracel- NHERF1 PDZ domains are necessary for the tumor lular cluster of is central to the assembly and suppressor effect. NHERF1 associates directly through the function of adherens junctions and the large complex is PDZ2 domain with b-catenin and is required for b-catenin composed of many components, of which b-catenin and localization at the cell–cell junctions in MEFs. Mechan- p120 catenin bind directly to the cytoplasmic tail of istically, the absence of NHERF1 selectively decreased cadherins (McCrea and Gumbiner, 1991; Reynolds the interaction of b-catenin with E-cadherin, but not with et al., 1994). b-catenin additionally acts as a transcrip- N-cadherin. The ensuing disorganization of E-cadherin- tional activator in the Wnt pathway (Nelson and Nusse, mediated adherens junctions as well as the observed 2004). Deregulation of Wnt signaling has been found in moderate increase in b-catenin transcriptional activity different types of cancer, in particular in colon cancer contributed most likely to the anchorage-independent where, most frequently, mutations in APC lead to growth of NHERF1-deficient MEFs. In vivo, NHERF1 is decreased protein turnover and cytoplasmic accumula- specifically localized at the apical brush-border membrane tion of b-catenin (Giles et al., 2003). Following in intestinal epithelial cells and is required to maintain translocation to the nucleus, b-catenin relieves T-cell a fraction of the cortical b-catenin at this level. Thus, factor/lymphoid enhancer factor (TCF/LEF)-mediated NHERF1 emerges as a cofactor essential for the inte- transcriptional repression to activate several oncogenes grity of epithelial tissues by maintaining the proper and other gene targets. Although the regulation of localization and complex assembly of b-catenin. b-catenin subcellular localization is a matter of extensive Oncogene (2007) 26, 5290–5299; doi:10.1038/sj.onc.1210336; investigation (Brembeck et al., 2004; Gottardi and published online 26 February 2007 Gumbiner, 2004), mechanisms of b-catenin shuttling between the membrane and cytoplasmic or nuclear Keywords: NHERF1/EBP50; b-catenin; E-cadherin; complexes are only beginning to be understood. transformation; colon cancer; mouse embryonic b-Catenin was shown to bind in vitro and in vivo fibroblasts (MEFs) through its C-terminal PDZ motif to Na/H exchanger regulator factor 1/ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (NHERF1/EBP50) (Shibata et al., 2003) that contains two PDZ domains and an ERM- binding region (Reczek et al., 1997; Weinman et al., Introduction 2003). NHERF1 is involved in the regulation of ion transporters and in the trafficking of many transmem- Transformed cells lack normal cell contact inhibition brane molecules (Shenolikar et al., 2004), but its role in and they grow in the absence of anchorage to the cancer is still controversial. NHERF1 gene mutations found with low frequency (3%) in breast cancer cell lines Correspondence: Dr M-M Georgescu, The University of Texas, and primary tumors were associated with loss of MD Anderson Cancer Center, 6767 Bertner Avenue, Houston, heterozygosity and increased aggressiveness (Dai et al., TX 77030, USA. E-mail: [email protected] 2004), suggesting a tumor suppressor role for NHERF1. Received 20 October 2006; revised 15 January 2007; accepted 15 January In contrast, Shibata et al. (2003) reported that the 2007; published online 26 February 2007 NHERF1/b-catenin complex promotes Wnt signaling NHERF1 stabilizes b-catenin/E-cadherin complexes EL Kreimann et al 5291 and may cooperate in the development of liver cancer. polygonal and more epithelial-like than their parental, To clarify the role of NHERF1, we analysed the growth non-immortalized counterparts (not shown). However, properties of mouse embryonic fibroblasts (MEFs) although immortalized NHERF1( þ / þ ) MEFs grew as generated from NHERF1-deficient mice (Morales et al., a monolayer on both plastic or poly-D-lysine-coated 2004). We found that NHERF1(À/À) immortalized glass surfaces, NHERF1(À/À) MEFs retracted and MEFs, in contrast to their wild-type counterparts, presen- clustered in spheres on poly-D-lysine-coated glass cover- ted anchorage-independent growth. In NHERF1 (À/À) slips (Figure 1a). Compared to NHERF1( þ / þ ) cells, cells, b-catenin was delocalized from the plasma mem- (À/À) MEFs presented delayed spreading when plated brane to the cytoplasm and formed weaker complexes on poly-D-lysine but not on collagen matrix (Figure 1b), with E-cadherin. Our results point to a tumor sup- or on fibronectin (not shown). Unlike collagen or pressor role for NHERF1 via stabilization of b-catenin/ fibronectin, poly-D-lysine promotes adhesion by an E-cadherin complexes at the plasma membrane. integrin-independent mechanism based on electrostatic interactions with the cell membrane, suggesting integrin- independent membrane modifications of NHERF1 Results (À/À) cells. Importantly, immortalized NHERF1(À/À) cells were able to form colonies in soft agar (Figure 1c), Loss of NHERF1 confers anchorage-independent growth suggesting that the loss of NHERF1 promotes ancho- to immortalized cells rage-independent growth. The analysis of the prolifera- To assess the role in tumor development of NHERF1, tion of parental and immortalized MEFs showed no we immortalized NHERF1( þ / þ ) and (À/À) MEFs significant difference between the parental counterparts with SV40T antigens and characterized their growth and a reproducible moderate proliferation advantage for properties. At confluence, immortalized MEFs appeared NHERF1(À/À) immortalized cells relative to ( þ / þ )

Figure 1 Anchorage-independent growth and increased migration of NHERF1(À/À) MEFs. (a) Immortalized NHERF1(À/À) cells grown on poly-D-lysine-coated glass coverslips formed cell spheres (arrows), whereas NHERF1( þ / þ ) grew as monolayer (not shown). (b) Immortalized MEFs were plated in triplicate on poly-D-lysine or collagen-coated glass coverslips and allowed to attach and spread for the indicated periods of time. Means7s.e.m. for cells not spread or spread were calculated from equivalent numbers of cells (N ¼ 50–80) counted on five fields per plate and were expressed as percentages. Significant differences between NHERF1( þ / þ ) and (À/À) cells are marked with asterisk (Po0.05). (c) Immortalized MEFs were examined for anchorage-independent growth by soft-agar assay. Colonies were visible for NHERF1(À/À) MEFs after 4 weeks of incubation. Images ( Â 100) were taken with a Zeiss Axiovert 200 microscope. The graph represents the number of colonies per plate calculated as mean7s.d. from triplicate plates. (d) Proliferation assay showing growth of parental and immortalized MEFs in DMEM containing 0.5% FBS. Cells were counted after 48 h incubation and values are expressed as means7s.d. from triplicate measurements. Significant differences are marked with asterisk (Po0.005). (e) Scratch-assay showing migration of immortalized MEFs on plastic culture dishes. Images ( Â 40) were taken at time 0 (0 h) showing the initial scratch of the monolayer and after 13 h of migration, when the NHERF1(À/À) cells closed the scratch (T ¼ 13 h). All these experiments were repeated three times with similar results.

Oncogene NHERF1 stabilizes b-catenin/E-cadherin complexes EL Kreimann et al 5292

Figure 2 Reconstitution of NHERF1 in NHERF1(À/À) cells suppresses colony formation. (a) Schematic organization of NHERF1 showing two PDZ domains and an EB region. NHERF1 FL and domain-deletion mutants are shown with amino-acid boundaries. (b–d) Immortalized NHERF1(À/À) MEFs were reconstituted by retroviral infection with NHERF1 FL and mutants and assayed for proliferation (b) and soft agar colony formation (c and d). The graph in (c) shows the average7s.d. of the number of colonies counted in triplicate plates. Significant differences, as compared to vector control, are marked with asterisk (Po0.005). Immunoblotting shows the levels of reconstituted proteins. These experiments were repeated three times with similar results.

cells (Figure 1d). We also investigated the mobility NHERF1 controls the intracellular distribution of MEFs by wound-healing assay. NHERF1(À/À) of b-catenin cells migrated faster than their ( þ / þ ) counterparts, NHERF1 is known to bind multiple ligands through the filling the scratch in 13 versus 16 h respectively PDZ1 domain, to NHE3 through both PDZ domains, (Figure 1e), suggesting cytoskeletal changes in and to b-catenin and Yap 65 specifically through the NHERF1(À/À) cells. PDZ2domain (Voltz et al., 2001; Weinman et al., 2003). Human NHERF1 and a panel of domain-deletion We confirmed in an overlay assay that b-Catenin binds mutants were reconstituted in NHERF1(À/À) immor- to NHERF1-PDZ2domain (Figure 3a). b-Catenin talized MEFs (Figure 2a). The proliferation rates of associated to overexpressed NHERF1 full-length and reconstituted cells and vector-control cells were found PDZ1-2mutant but not to the I2isoform (Figure 3b). similar (Figure 2b). These cells were thus tested for The failure of this PDZ2-deleted isoform to suppress their ability to form colonies in soft agar (Figure 2c anchorage-independent growth (Figure 2c and d) raised and d). Full-length NHERF1 suppressed the colony the possibility that b-catenin might contribute to formation of NHERF1(À/À) cells, supportive of a NHERF1’s growth suppression. We therefore examined tumor suppressor function for NHERF1. To the same the subcellular distribution of b-catenin in NHERF1 extent, the mutant PDZ1-2, which lacks the ERM- ( þ / þ ) and (À/À) immortalized MEFs. Immunofluor- binding (EB) region, suppressed colony formation, escence analysis showed strong b-catenin staining at the whereas all the other mutants had no effect or slightly membrane boundaries between adjacent NHERF1 increased growth in soft agar. Notably, the naturally ( þ / þ ) cells but not of NHERF1(À/À) cells (Figure 3c). occurring splice isoform I2that lacks the PDZ2 Furthermore, the fractionation of cellular proteins indica- domain (Morales et al., 2004) was unable to suppress ted a marked increase of b-catenin in the cytoplasmic growth, indicating that both PDZ domains are fraction of NHERF1(À/À) cells (Figure 3d). Although required for anchorage-independent growth suppres- we could not detect nuclear b-catenin by immuno- sion by NHERF1. fluorescence analysis, we measured the transactivation

Oncogene NHERF1 stabilizes b-catenin/E-cadherin complexes EL Kreimann et al 5293

Figure 3 Altered intracellular distribution of b-catenin in NHERF1(À/À) MEFs. (a) Overlay assay of filter-immobilized purified proteins (0.5 mg), showing interaction between NHERF1 FL or PDZ2-EB and overlaying Myc-tagged GST-b-catenin C terminus (10 mg/ml). Bound b-catenin was revealed by immunoblotting the membrane with Myc antibody. The membrane was stripped and probed with GST antibody to visualize the immobilized NHERF1 constructs. (b) Precipitation (IP) with GST-b-catenin of NHERF1 proteins overexpressed in OKP cells from pCMV vector. Total cell lysates (TL) show equal amounts of input NHERF1 proteins and GST-b-Catenin. (c) Immunofluorescence analysis of b-catenin showing membrane localization at cell boundaries in NHERF1( þ / þ ) immortalized MEFs. Cells fixed with 3% formaldehyde were stained for b-catenin (red) and nuclei (DAPI, blue). Images ( Â 200) were taken with a Zeiss Axioskop 40 microscope. b-catenin staining at the cell–cell boundaries was quantified by ImageJ software that measured the total length of visible cell contacts (in pixels). Average7s.d. were calculated from three images per cell type, each containing 30–40 cells. (d) Subcellular fractionation of immortalized MEFs into nuclear (N), cytoplasmic (C), membrane (M), cytoskeleton (Cs) and debris (D) fractions. By using this procedure, it is known that the nuclear fraction can be contaminated by membrane components and vice versa, whereas the cytoplasmic fraction is pure. This is shown here by using PARP as nuclear marker. The ratio between NHERF1(À/À) and ( þ / þ ) levels of b-catenin normalized to actin levels in the various fractions was graphically represented. The results are represented as average7s.d. from three experiments using two different immortalized MEF populations. Note high levels of b-catenin in the cytoplasmic fractions of NHERF1(À/À) MEFs (Po0.001). (e) Luciferase reporter activity assay showing increased transcriptional activity of b-catenin in NHERF1(À/À) MEFs. Values represent the ratio of TOP versus FOP firefly luciferase activities normalized previously to renilla luciferase activity. The average7s.d. were calculated from triplicate plates and experiments were repeated three times with similar results.

potential of endogenous b-catenin by using a luciferase examined their growth in soft agar. Vector control and reporter TOP-FOP assay (Figure 3e). We found N-cadherin-CT expressing NHERF1(À/À) cells formed moderate increase of b-catenin transactivation ability equivalent numbers of colonies in soft agar. Never- in NHERF1(À/À) cells, indicating the presence of an theless, N-cadherin-CT expression significantly reduced increased nuclear pool of b-catenin in these cells. the size of the colonies (Figure 4), indicating that Expression of a short C-terminal fragment of N- b-catenin transactivation contributes to the anchorage- cadherin that encompasses the binding site for b-catenin independent growth of NHERF1(À/À) MEFs. was shown to couple b-catenin in the cytoplasm and to In vivo, we have described previously structural prevent b-catenin entry and transactivation in the defects of the brush-border membrane (BBM) of nucleus (Sadot et al., 1998). We stably expressed this intestinal epithelial cells in NHERF1(À/À) mice N-cadherin-CT fragment in NHERF1(À/À) MEFs and (Morales et al., 2004). Fractionation of intestinal BBM

Oncogene NHERF1 stabilizes b-catenin/E-cadherin complexes EL Kreimann et al 5294

Figure 4 N-cadherin C-terminal fragment partially suppressed the anchorage-independent growth of NHERF1(À/À) MEFs. Schematic representation of N-cadherin extracellular and intracellular domains and of the C-terminal (CT) fragment that binds to b-catenin. N-cadherin CT fragment or retroviral vector control was expressed in immortalized NHERF1(À/À) MEFs. Cells were assayed for anchorage-independent growth by soft agar assay as in Figure 1c. The size of colonies was measured (legend) and the size distribution was graphically represented as percentage of total number of colonies. Significant differences between vector and N-cadherin-CT-expressing cells were obtained for the development of small size colonies (Pp0.005) and large size colonies (Pp0.01). This experiment was repeated twice with similar results.

from NHERF1( þ / þ ) and (À/À) mice revealed lower cells, we expressed short hairpin (sh) inhibitory RNA b-catenin levels in the BBM fractions of mutant mice against NHERF1 in the human colon SW480 (Figure 5a). Immunofluorescence analysis of intestinal cells that contain elevated NHERF1 levels and obtained epithelial cells showed decreased b-catenin apical approximately 50% depletion of NHERF1 (Figure 6e). membrane staining in NHERF1(À/À) mice (Figure 5b Reciprocal coimmunoprecipitations of b-catenin and and c). In contrast, no difference of cytokeratin E-cadherin showed weakened interaction between intracellular localization was apparent in wild-type or b-catenin and E-cadherin in NHERF1-depleted cells mutant mice. In NHERF1( þ / þ ) mice, b-catenin compared with control cells (Figure 6f). colocalized with NHERF1 at the BBM, suggesting that NHERF1 stabilizes b-catenin at the apical membrane in intestinal epithelial cells. Decreased E-cadherin cell surface levels in NHERF1(À/À) cells The decrease of E-cadherin from the plasma membrane Decreased association of b-catenin with E-cadherin in the is a marker of epithelial cancer progression and absence of NHERF1 correlates with cell transformation and increased To determine whether the reduced plasma membrane invasiveness (Thiery, 2002). We examined the levels of distribution of b-catenin in NHERF1(À/À) MEFs E- and N-cadherin from total cell lysates in both reflected a decrease of b-catenin from the catenin– parental and immortalized MEFs. In general, parental cadherin complexes, we immunoprecipitated E-cadherin MEFs presented similar levels of N-cadherin but lower (Figure 6a) and N-cadherin (Figure 6b) and found levels of E-cadherin compared to matched immortalized reduced association of b-catenin with E-cadherin but MEFs (Figure 7a). In both parental and immortalized not with N-cadherin in NHERF1(À/À) cells. Similar MEFs, the absence of NHERF1 determined opposite results were obtained by reciprocal coimmunoprecipita- variations of the total expression levels of cadherins, tions with b-catenin that coupled equivalent amounts of with increase in N-cadherin and decrease in E-cadherin N-cadherin but lower amounts of E-cadherin, a-catenin (Figure 7a). These variations of cadherin levels were not and p120 catenin in NHERF1(À/À) versus ( þ / þ ) caused by changes in transcription, as determined by immortalized MEFs (Figure 6c). Moreover, coimmuno- RT–PCR (not shown). precipitation of p120 catenin and a-catenin with The immunofluorescence analysis showed in immor- N-cadherin showed equal amounts in both cell types, talized wild-type cells E-cadherin localization at the indicating that the catenin complexes formed with boundaries between adjacent cells in patches corre- N-cadherin are unchanged by NHERF1 (Figure 6b). sponding to punctate adherens junctions. In contrast, in To confirm that NHERF1 is responsible for increased NHERF1(À/À) cells, E-cadherin staining was peri- association between b-catenin and E-cadherin, the nuclear, being absent at cell–cell contacts. N-cadherin complex formation was analysed in NHERF1(À/À) appeared at the cell boundaries in both NHERF1 immortalized MEFs reconstituted with NHERF1 full- ( þ / þ ) and (À/À) cells, with stronger staining at length, I2isoform or vector control. Cell reconstituted punctate adherens junctions in NHERF1(À/À) cells. with NHERF1 full-length, but not with I2or vector The quantification of cell surface protein levels by showed higher amounts of E-cadherin coimmunopreci- biotinylation showed significantly decreased E-cadherin pitated with b-catenin (Figure 6d). The amounts of levels, moderately decreased b-catenin levels and un- N-cadherin coimmunoprecipitated with b-catenin were changed N-cadherin levels in NHERF1(À/À) versus equivalent in all cell types (Figure 6d), indicating ( þ / þ ) immortalized MEFs (Figure 7c). In parental a preferential stabilization of b-catenin/E-cadherin cells, N-cadherin and b-catenin surface levels presented complexes by NHERF1. opposite variations, with more N-cadherin and less To confirm the importance of NHERF1 for b-catenin in NHERF1(À/À) (Figure 7d). Altogether, b-catenin/E-cadherin complex formation in mammalian these findings showed that loss of NHERF1 decreased

Oncogene NHERF1 stabilizes b-catenin/E-cadherin complexes EL Kreimann et al 5295

Figure 5 Decreased b-catenin levels in small intestine BBM from NHERF1 (À/À) mice. (a) Fractionation of intestinal BBM fractions (P3, P4) from NHERF1 ( þ / þ ) and (À/À) mice showing decreased levels of b-catenin in NHERF1(À/À) mice. Full-length NHERF1 (arrowhead) and low-molecular-weight cleavage fragments (bracket) are shown. b-Catenin expression was normalized to vinculin and represented graphically. This experiment was performed three times with similar results. (b) Immunofluorescence analysis of small intestine epithelial cells with b-catenin (left), NHERF1 superposed on DIC (middle) and cytokeratin (right) antibodies. ToPro-3 iodide was used for nuclear staining. Arrowheads indicate the BBM. Images ( Â 630) were taken with a Zeiss LSM 510 confocal microscope. (c) The intensity of apical and lateral b-catenin membrane staining was measured by using the Metamorph software and the apical/ lateral intensity ratio was represented for individual cells (left graph) and as average7s.d. of individual measurements (right graph). both b-catenin and E-cadherin levels from the plasma Using reconstitution experiments of various NHERF1 membrane, whereas N-cadherin levels either remained domains in NHERF1-deficient cells, we found that both unchanged or increased. PDZ domains are required for growth suppression. In contrast to the PDZ1 domain that accounts for the majority of NHERF1 heterotypic associations, the Discussion PDZ2domain interacts specifically with only few partners that include b-catenin (Shibata et al., 2003). The importance of NHERF1 for ion transporter Involved extensively cancer progression, especially of regulation, stabilization of transmembrane receptors to colon carcinoma, b-catenin engages in various protein the plasma membrane and organization of the epithelial complexes in membrane, cytoplasmic and nuclear apical microvilli is well documented (Morales et al., compartments (Nelson and Nusse, 2004). This cellular 2004; Shenolikar et al., 2004). These functions rely on compartmentalization dictates b-catenin’s functions, as the interaction of NHERF1 with transmembrane a tumor suppressor at the membrane where it interacts partners through the PDZ domains and with ERM with E-cadherin and stabilizes adherens junctions, or as proteins through the EB region. Recently, a role for an oncogene in the nucleus where, following Wnt NHERF1 in cancer emerged but it is still not clear signaling or perhaps release from the membrane, whether as an oncogene or as a tumor suppressor complexes with TCF/LEF transcription factors and (Shibata et al., 2003; Dai et al., 2004). We show here transactivates including oncogenes (Giles et al., that NHERF1 acts as a tumor suppressor and is 2003). We found that NHERF1 is necessary for b- required to suppress anchorage-independent growth. catenin stabilization at the plasma membrane. In MEFs,

Oncogene NHERF1 stabilizes b-catenin/E-cadherin complexes EL Kreimann et al 5296 membrane with compensatory intestine elongation or from b-catenin redistribution from the apical membrane to the cytoplasm and nucleus with increased Wnt/ b-catenin pathway activation and intestinal cell proliferation. Both parental and immortalized NHERF1(À/À) MEFs had E-cadherin levels significantly reduced compared to NHERF1( þ / þ ) MEFs. In contrast, the N-cadherin levels slightly increased in NHERF1(À/À) cells, reminiscent of the process of epithelial to mesenchymal transition that leads to mesenchymal appearance and increased motility of epithelial cells (Thiery, 2002). Although the appearance of the cells was not modified, NHERF1(À/À) cells migrated faster than their wild-type counterparts and clustered in spheres, suggesting loss of cell–cell contact inhibition. NHERF1(À/À) immortalized MEFs also proliferated more than their wild-type counterparts. This prolifera- tive advantage might be caused in part by the enhance- ment of b-catenin’s transactivation ability, but probably also by loss of NHERF1 interactions with other binding partners. In this respect, we reported that NHERF1 attenuates the PI3K growth promoting signaling down- stream of PDGFR by an interaction between its PDZ1 domain and PTEN tumor suppressor (Takahashi et al., 2006). A simplified mechanistic model of how mem- brane-localized NHERF1 might suppress tumor growth is presented in Figure 8. Pharmacological inhibition of PDGFR signaling (Supplementary Figure 1) as well as inhibition of b-catenin transactivation by a dominant- negative N-cadherin fragment impaired the anchorage- independent growth of NHERF1(À/À) MEFs, arguing Figure 6 Decreased binding of b-catenin to E-cadherin for NHERF1’s growth suppression via multiple path- in NHERF1(À/À) MEFs and SW480 cells. Co-immunoprecipitation ways downstream of signaling proteins that interact (IP) of endogenous b-catenin with E-cadherin (a) or with N- with its PDZ domains. cadherin (b) and reciprocal co-immunoprecipitation of E-cadherin, NHERF1 deficiency decreased the interaction be- p120, a-catenin and N-cadherin with b-catenin (c) from cell lysates of confluent immortalized MEFs. Matched rabbit (r) and mouse tween b-catenin and E-cadherin in MEFs and in colon (m) IgG were used as controls for immunoprecipitation. Immuno- carcinoma SW480 cells but had no effect on the blotting (IB) was performed with the indicated antibodies. TL, b-catenin/N-cadherin complex. These observations sug- total cell lysate. (d) NHERF1(À/À) immortalized MEFs recon- gested that although b-catenin formed a stable complex stituted with NHERF1 FL, I2or vector were lysed and endogenous E-cadherin and N-cadherin were co-immunoprecipitated (IP) with with N-cadherin, it required association with NHERF1 b-catenin. (e) Depletion of NHERF1 by shRNA (sh#1) in SW480 to form a stable complex with E-cadherin. We do not cells. Cells containing NHERF1 sh#1 or control (Ctr) shRNA were know yet the mechanism by which NHERF1 selectively lysed after 5 days of drug selection. NHERF1 and b-catenin stabilized the b-catenin/E-cadherin complexes. expression levels were normalized to actin and represented NHERF1 was shown to stabilize at the cell surface graphically. (f) Co-immunoprecipitation (IP) with indicated anti- bodies and controls (top) of b-catenin and E-cadherin from SW480 transmembrane proteins with which it directly asso- cells treated with control (Ctr) or NHERF1 shRNA (sh#1). The ciated either by decreasing their endocytosis or by levels of associated E-cadherin normalized to immunoprecipitated increasing their recycling to the plasma membrane b-catenin (left) and of associated b-catenin normalized to (Shenolikar et al., 2004). Here, NHERF1 might stabilize immunoprecipitated E-cadherin (right) were quantified and showed decrease in NHERF1-depleted cells compared to control cells. E-cadherin by what appears to be an indirect mechan- ism involving interaction with b-catenin. It is concei- vable that NHERF1 inhibits the endocytosis of the b-catenin/E-cadherin complex. Alternatively, as mutagen- NHERF1 deficiency impaired the localization of esis of the b-catenin-binding site prevented E-cadherin b-catenin at the cell–cell junctions. In intestinal epithe- processing and transport to the plasma membrane lial cells where NHERF1 is localized at the apical BBM, (Chen et al., 1999), NHERF1 could influence the b-catenin was specifically reduced apically in NHERF1 b-catenin/E-cadherin complex assembly and trafficking (À/À) mice. A macroscopic defect of NHERF1(À/À) from ER to the plasma membrane. As cadherin switch- mice is the increased length of the small intestine (data ing is now well accepted to play a central role in both not shown). We currently examine whether the increased developmental and pathological conditions (Takeichi, intestine length could result from defects of the apical 1995; Thiery, 2002), further studies will address the

Oncogene NHERF1 stabilizes b-catenin/E-cadherin complexes EL Kreimann et al 5297

Figure 7 Decreased membrane localization of E-cadherin in NHERF1(À/À) MEFs. (a) Total cell lysates (50 mg proteins) from confluent parental (Par.) and immortalized (Immort.) MEFs were immunoblotted with the indicated antibodies. Note opposite variations of E- and N-cadherin levels, which were normalized to actin levels and quantified in the graphs. (b) Immunofluorescence analysis of methanol–acetone-fixed immortalized MEFs labeled with indicated antibodies and DAPI (blue). Image magnification ( Â 400). (c and d) Biotinylation of plasma membrane proteins from immortalized (c) or parental (d) MEFs. Biotinylated proteins were precipitated with streptavidin (SA)-agarose beads and immunoblotted with indicated antibodies (left). IGF1Rb and Erk2were used as membrane and cytoplasmic markers, respectively. The graph shows the cell surface levels of biotinylated proteins normalized to IGF1Rb surface levels. mechanism of differential regulation of E-cadherin (ACCCCATCCTAGACTTCAA) and control EGFP shRNA versus N-cadherin levels and complexes by NHERF1. (gift from Tsuyoshi Akagi) were cloned in pSIREN-RetroQ This will clarify the mechanism by which NHERF1 vector. modulates cell adhesion and motility with impact on both normal development and cancer progression. Cells, retroviral infection, soft agar, cell spreading and migration assays Bosc23, SW480 and MEFs were grown in DME medium. Materials and methods Opossum kidney (OKP) cells (gift from Patricia Preisig) were grown in 1:1 mixture of low glucose DMEHam’s F12media. Plasmids Retroviral infections and soft-agar assays were described For MEF immortalization, the SV40-T-antigens (gift from (Georgescu et al., 1999). For spreading assays, 5 Â 104 cells Tomoyuki Shishido) were used as described (Suzuki et al., were plated on coverslips coated with poly-D-lysine (Becton 2004). Human NHERF1 full-length (FL) and deletion mutants Dickinson, Franklin Lakes, NJ, USA) or 0.5 mg/ml collagen I2, PDZ1iP, PDZ2, PDZ2-EB and PDZ1-2 were cloned in (Inamed Biomaterials, Fremont, CA, USA). After incubation, pCXb retroviral vector and in pGEX-6P-1 vector for the cells were fixed with 4% formaldehyde for 30 min. Cells recombinant protein expression. A b-catenin fragment encod- were scored as spread when extensions of the membrane were ing the last 100 amino acids was inserted into pGEX-Myc visible and not spread when they were attached with no visible vector. An N-cadherin fragment encoding the last 70 residues membrane protrusion. For wound-healing assay, a MEF was inserted into pCXb-Myc vector. NHERF1 shRNA#1 monolayer was scratched with a pipette tip in DME medium

Oncogene NHERF1 stabilizes b-catenin/E-cadherin complexes EL Kreimann et al 5298 buffer volumes (50 mM HEPES, pH 7.9, 250 mM KCl, 0.1% NP40, 0.1 mM (ethylene glycol bis(b-aminoethylether)- N,N,N0,N0,-tetraacetic acid), 0.1 mM EDTA and 10% glycerol) and centrifuged (16 000 g, 10 min). The resulting supernatant corresponded to the nuclear fraction and the pellet to the debris. All procedures were performed on ice and all buffers contained protease and phosphatase inhibitors (Georgescu et al., 1999). The BBM purification was performed as described previously (Morales et al., 2004).

Biotinylation of membrane proteins Confluent cultures were washed three times with ice-cold phosphate buffered saline PBS (pH 8) and biotinylated on ice with 0.5 mg/ml EZ-Link Sulfo-NHS-Biotin (Pierce, Rockford, IL, USA) in PBS for 30 min. The biotin excess was washed twice with ice-cold PBS containing 100 mM glycine. The cells were lysed, and 0.5 mg of proteins was incubated with streptavidin immobilized on Agarose CL-4B beads (Biochem- Figure 8 Simplified model of NHERF1 role as tumor suppressor. 1 NHERF1 could influence cell growth by interacting with many ika, St Louis, MO, USA) overnight at 4 C. ligands (reviewed in Voltz et al. (2001)). Interactions that suppress cell growth include but are not limited to those with PTEN through Immunofluorescence analysis the PDZ1 domain, with b-catenin through the PDZ2domain and with the ERM-neurofibromatosis type 2(NF2)proteins through Immunofluorescence analysis of MEFs plated on poly-D- the EB region. Dashed lines indicate putative effects. lysine-coated coverslips was performed as described previously (Takahashi et al., 2006). For mouse intestine, paraffin blocks were prepared from 9-week-old NHERF1( þ / þ ) and (À/À) littermates and stained as described previously (Morales et al., containing 0.5% fetal bovine serum (FBS) and 5 mg/ml 2004). Antibodies are specified in Supplementary material. mitomycin C (Sigma, St Louis, MO, USA) to prevent proliferation during migration. Luciferase assay Immortalized 5 Â 104 MEFs per well were serum-deprived and Cell lysis and protein analysis cotransfected with 0.5 mg of either TOP or FOP luciferase (gift Cell lysis, overlay assay, immunoprecipitation and immuno- from Barry Gumbiner) and 0.05 mg of renilla luciferase using blotting were performed as described previously (Georgescu 2 ml/well Lipofectamine 2000 (Invitrogen, Carlsbad, CA, et al., 1999; Takahashi et al., 2006). Antibodies are specified in USA). After 48 h, the cells were prepared using the Dual Supplementary material. For cell fractionation, 4 Â 106 cells Luciferase Reporter assay system (Promega, Madison, WI, were homogenized in 1 ml of hypotonic buffer (10 mM HEPES, USA), according to the manufacturer’s instructions. pH 7.5, 10 mM KCl, and 3 mM MgCl2). After centrifugation (600 g, 5 min), the supernatant SN1 was centrifuged (100 000 g, 1 h) and the resulting SN2represented the cytoplasmic Acknowledgements fraction. The pellet P2was resuspended in membrane solubilization buffer (10 mM Tris–HCl pH 7.4, 500 mM NaCl, MMG acknowledges support from MD Anderson Cancer 1mM ethylenediaminetetraacetic acid (EDTA) and 1% Triton Center Tobacco Fund and NCI-CA107201, FCM from X-100) and centrifuged (16 000 g, 30 min). The supernatant S3 American Brain Tumor Association and ELK from corresponded to the membrane fraction and the pellet P3 to NCI-CA09299-26. NCI-CA16672 partially supported animal the cytoskeleton. The initial pellet P1 was resuspended in 10 breeding.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene