- Email: [email protected]
β-catenin signaling activity and migration capacity in human colon carcinoma cells
Biochemical and Biophysical Research Communications 380 (2009) 478–483
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Smad4 restoration leads to a suppression of Wnt/b-catenin signaling activity and migration capacity in human colon carcinoma cells Xiaoxiao Tian a, Hao Du b, Xiangsheng Fu a, Kang Li a, Aimin Li a, Yali Zhang a,* a b
Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou Road 1838, Guangzhou 510515, Guangdong Province, China Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, China
a r t i c l e
i n f o
Article history: Received 15 January 2009 Available online 25 January 2009
Keywords: Smad4 Tumor-suppressor gene Beta-catenin E-cadherin Colon cancer
a b s t r a c t Recent studies have reported that Smad4 has a TGF-b-independent function as a tumor suppressor in cooperating with b-catenin/Lef to regulate target gene expression. The objective of this research was to study the role of Smad4 in colon cancer migration and its potential mechanism. In our study, stable colon cancer cells that have increased Smad4 expression were created using a eukaryotic vector containing Smad4-pcDNA3.1(+). Smad4 restoration directly suppressed the Wnt/b-catenin signal activity in SW480 cells by down-regulating b-catenin expression and altering its localization from cytoplasm and nucleus to the plasma membrane. Up-regulation of Smad4 also increased the expression of E-cadherin and decreased the transcriptional activity of b-catenin/Tcf target genes, such as claudin-1 and MMP-7. SW480 cells with increased Smad4 expression had decreased in vitro cell migration. The data suggested that restoration of Smad4 in Smad4-deficient cells may provide a potential therapeutic strategy for intervention of colon cancer migration and metastasis. Ó 2009 Elsevier Inc. All rights reserved.
Smad4, originally characterized as a central intracellular signal transduction component of the transforming growth factor b (TGFb) family in epithelial cells, is regarded as a tumor suppressor gene predominantly involved in gastrointestinal carcinogenesis [1]. Loss of Smad4 function either due to loss of its expression or genetic mutation is considered to be a genetically late step and occurs in a significant proportion of colon and pancreatic cancer [2]. The Wnt/b-catenin signaling is implicated in colon carcinogenesis and suppression of this signal pathway is associated with a reduction of cellular proliferation and the induction of cellular differentiation [3]. b-catenin performs dual functions, which include a crucial role in cell–cell adhesion and the Wnt/b-catenin pathway [4]. Activation of Wnt signaling inhibits GSK dependent phosphorylation of b-catenin and results in an increase in b-catenin protein levels. The accumulation of b-catenin promotes its nuclear translocation where it regulates Wnt target genes transcription in collaboration with factors from the Tcf/Lef family [5]. Both TGFb and Wnt signaling pathways are thought to play critical roles in regulating the differentiation processes of colonic epithelial cells. Increasing evidence of cross talk between Smad signaling and the Wnt pathway has been reported during the past years [6,7]. Nevertheless, the relationship between the migrationsuppressive responses of Smad4 and the activity of Wnt signaling pathway is not thoroughly understood. Recent evidences have suggested additional mechanisms underlying the effects of Smad4, for * Corresponding author. Fax: +86 20 87280770. E-mail address: [email protected] (Y. Zhang). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.01.124
example, re-expression of Smad4 in the Smad4-deficient SW480 colon carcinoma cells resulted in loss of tumorigenicity of these cells in nude mice that was accompanied by the restoration of a more epithelioid morphology and induced transcription of E-cadherin and P-cadherin [8], in addition, it was recently shown that a novel mechanism underlying Smad4 invasion-suppressive function through regulation of a potential metastatic modulator, claudin-1, in a TGFb-independent manner [9]. Cell–cell adhesion in epithelial cell sheets is maintained mainly through adherens junctions and tight junctions [1]. E-cadherin is a key player in cell–cell adhesion connecting adjacent cells via the cadherin–catenin adhesion complex. Claudin-1, the most apical cell–cell contacts and the most important tight junction for barrier function in colon cancer, was recently identified as a target of Wnt/b-catenin signaling [10]. Based on these studies, we hypothesized that there was a relationship between Smad4, a tumor suppressor protein, and Wnt/b-catenin signaling, a progression-promoting pathway, in invasive and migratory colorectal carcinoma. The objective of this study was to investigate the impact of enhanced Smad4 expression on b-catenin and E-cadherin protein expression and localization in colon cancer cells and its effect on cell migration. In this study, we reported that Smad4 directly suppressed the Wnt/b-catenin signal pathway in Smad4-responsive human colon carcinoma cells by down-regulating the cytoplasmic and nucleus expression of b-catenin and increasing localization of b-catenin to the plasma membrane. We further showed that Smad4 induced the expression of the intercellular adhesion molecule E-cadherin and reduced transcriptional activity of b-catenin/
X. Tian et al. / Biochemical and Biophysical Research Communications 380 (2009) 478–483
Tcf target genes, such as claudin-1 and MMP-7. Lastly, we found that the above-mentioned reasons contributed to the migrationinhibitory effect of Smad4 in colon cancer cells. Materials and methods Cell cultures, plasmids and antibodies. Human colon carcinoma SW480 cells was obtained from ATCC and cultured in RPMI 1640 media supplemented with 10% fetal bovine serum. The full-length coding sequence of Smad4 was constructed into the eukaryotic expression vector pcDNA3.1(+) for stable transfection. The sequence of the recombinant plasmid was confirmed by sequencing. Empty vector pcDNA3.1(+) was used as control. The Lef/Tcf reporter plasmid, TOPflash, and its mutant control, FOPflash, were generously provided by Dr. Xinying Wang (Nanfang Hospital, Guangzhou, China). Primary immunoblotting antibodies were: anti-claudin-1 (Cell Signaling Technology, Beverly, MA), anti-Smad4, anti-b-catenin, anti-E-cadherin and anti-b-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA). All secondary antibodies used were obtained from Santa Cruz Biotechnology Inc. Overexpression of Smad4 by stable transfection. One day before transfection, the SW480 cells were seeded in 6-well cell culture plates to provide a final density of 60–70% confluence. Cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and selected in complete medium containing 1mg/ml G418 sulfate (Gibco BRL, USA) at 48h after transfection, G418-resistant cell clones were isolated for 2 weeks. After expansion of each individual clone, cells were screened for expression of Smad4 by immunoblot analysis. Two clones that expressed similar levels of Smad4 in protein concentrations were selected for subsequent experiments. SW480 cells that were stably transfected with pcDNA3.1(+) were used as a control. Immunoblot analysis. Cultured and harvested cells were lysed in RIPA buffer supplemented with protease inhibitors. Lysates were centrifuged at 15,000g for 30 min at 4 °C. To ascertain b-catenin cellular localization, cytoplasmic and nuclear proteins were isolated from cells using a cell fractionation kit (Keygen, NanJing, China). Equal amounts of protein samples were electrophoretically separated by SDS–PAGE in separation gels and transferred to nitrocellulose sheets. They were blocked in TBS-T containing 5% non-fat dry milk and then incubated overnight at 4 °C with the primary antibodies diluted in the same buffer (Smad4 1:400; E-cadherin 1:600; b-catenin 1:1000; claudin-1 1:1000; b-actin 1:3000 dilutions). Membranes were washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000 dilutions) for enhanced chemiluminescence detection using an ECL detection kit (Pierce Biotechnology Inc., Rockford, IL). Semiquantitative RT-PCR. Total RNA was isolated from the cell lines using the TRIzol reagent (Invitrogen, Carlsbad, CA). Reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit(Fermentas), 2 lg RNA as template and oligo(dT) as primer. PCR analysis was performed using gene-specific primers and diluted first-strand cDNA as template. Primers for Smad4: Forward 50 -GTGGCTGGTCGGAAAGGATT-30 , Reverse 50 -ACTGGCA GGCTGACTTGTGG-30 ; b-catenin: Forward 50 -ATCATCGTGAGGGCTT ACTG-30 , Reverse 50 -CCATCCCTTCCTGTTTAGTT-30 ; claudin-1: Forward 50 -GCCCTACTTTGCTGTTCCTG-30 ; Reverse 50 -CCCTTCCCCCAAT TGAGTAT-30 ; MMP-7: Forward 50 -TGGAGTGCCAGATGTTGCAG-30 , Reverse 50 -TTTCCATATAACTTCTGAATGCCT-30 ; GAPDH: Forward 50 -CGGGAAGCTTGTCATCAATGG-30 , Reverse 50 -GGCAGTGATGG CATGGACTG-30 . Immunofluorescence. Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% TritonX-100 solution and blocked in 3% bovine serum albumin–0.2% Triton X-100. Samples were then incubated with the following primary antibodies:
479
anti-b-catenin (mouse monoclonal); anti-claudin-1 and -E-cadherin (rabbit polyclonal). FITC anti-mouse and anti-rabbit, Rhodamine Red-X anti-rabbit were used as secondary antibodies at 1:200 dilutions, respectively. Stained glass coverslips were viewed and photographed using an OLYMPUS fluorescence microscope. Transient transfection and luciferase assays. For transient transfection assays, cells were seeded in 24-well plates in triplicate. Cells were cotransfected with a firefly luciferase reporter construct TOPflash or FOPflash, and a reference construct that contains Renilla reniformis luciferase, pRL-SV40 (Promega, Madison, WI), using Lipofectamine 2000. Forty-eight hours later, luciferase activities were measured using the Dual-Luciferase Reporter Assay System Kit (Promega) in a GloMax Luminometry System (Promega). Firefly luciferase activity was normalized to Renilla reniformis luciferase activity and plotted as mean ± SD from three independent experiments. Boyden chamber migration assay. Cells were resuspended in 0.2% bovine serum albumin serum-free medium at a concentration of 5 105 cells/ml, and 0.2 ml of each was seeded in the upper compartment of the chamber (24-well insert, 8 lm pore size; Millipore, Bedford, MA). About 0.6 ml appropriate medium supplemented with 10% fetal bovine serum was added to each well of the plate in the lower chamber. After 24 h of incubation, cells remaining inside the inserts were removed with cotton swabs, those that had migrated to the lower surface were fixed in formaldehyde and stained with crystal violet. Cells were counted in 5 random fields and expressed as the average number of cells per field under a light microscope. Each invasion assay was done in triplicate and repeated thrice. Results Smad4 restoration decreases b-catenin expression and alters its localization SW480 human colon carcinoma cells are Smad4-deficient, they lost one copy of chromosome 18, where Smad4 gene is located, the second Smad4 allele harbors a splice site mutation and is not expressed [11]. As shown in Fig. 1A and B, two clones of SW480 with stable expression of Smad4 were established using Smad4 eukaryotic expression plasmid. This cell line was established so that the effects of Smad4 re-expression on Wnt signaling activity could be compared. To determine whether Smad4 regulates b-catenin protein or mRNA expression, we prepared total RNA and cytoplasmic and nuclear proteins from parental, pc-SW480 (expressing empty vector) and Smad4-SW480 (overexpressing Smad4) cells, respectively, then subjected them to semiquantitative RT-PCR and immunoblot analysis as described in Methods. We detected a 40% reduction in the levels of cytoplasmic b-catenin and 60% in the nucleus in Smad4-SW480 clones when compared with SW480 cells (Fig. 1B). A 40% reduction in b-catenin mRNA expression was observed in Smad4-transfected cells (Fig. 1A). As the function of bcatenin in signal transduction depends on its cytoplasmic/nuclear localization, we further analyzed the distribution of the b-catenin protein through indirect immunofluorescence. SW480 cells showed strong staining for b-catenin in the cytoplasm and nucleus. Smad4 re-expression cells, in contrast, showed staining in the plasma membrane. The data demonstrated that up-regulation of Smad4 caused the relocalization of b-catenin from cytoplasm and nucleus to the plasma membrane in SW480 cells (Fig. 2B). Re-expression of Smad4 in SW480 cells leads to a reduction in bcatenin/Tcf signaling The presence of low cytoplasmic and nuclear levels of b-catenin suggested that Wnt/b-catenin signaling was impaired in Smad4SW480 cells. To explore the possibility, we analyzed b-catenin/
480
X. Tian et al. / Biochemical and Biophysical Research Communications 380 (2009) 478–483
Fig. 1. SW480 cells with increased Smad4 expression and its effect on b-catenin expression. Cells were transfected with recombinant plasmid Smad4-pcDNA3.1(+) and two Smad4-SW480 clones were obtained. (A) Total RNA from the indicated cells were isolated as described in Methods and subjected to RT-PCR for Smad4 and b-catenin. The results were normalized to GAPDH and presented as means ± SD of three independent experiments. There was less b-catenin mRNA expression in Smad4-transfected cells than Smad4-negative colon carcinoma cells (p < 0.05). (B) Immunoblot analysis for the expression of Smad4 and b-catenin in SW480, pc-SW480 and two Smad4-SW480 clones, b-actin was used as a control for protein loading. The E-cadherin protein expression levels were normalized to b-actin and the results were presented as means ± SD of three independent experiments. Relatively less b-catenin was detected in the cytoplasmic and nuclear fractions of Smad4-transfected cells compared with SW480 and pcSW480 groups.
Tcf signaling activity in SW480, pc-SW480 and Smad4-SW480 cells by transiently transfecting TCF and mutant TCF reporter plasmids (TOPflash and FOPflash, respectively). To control for transfection efficiency, we cotransfected a pRL-SV40 reporter construct as internal control. Relative luciferase activities were calculated as described in Methods. As shown in Fig. 2A, the relative transcriptional activity of the b-catenin/Lef complex was decreased (3- to 4-fold) in Smad4-SW480 cells compared with SW480 or pcSW480 cells. This analysis confirmed that Wnt signaling activity was significantly suppressed by Smad4 expression.
expression changed the localization of E-cadherin and claudin-1. The localization of E-cadherin and claudin-1 was not altered in Smad4-SW480 cells, however, restoration of Smad4 resulted in increased E-cadherin expression at the cell membrane and reduced claudin-1 expression in the cytoplasm (Fig. 3B). The observations were coincident with immunoblot analysis of the two proteins. The results of these two studies indicated a decrease in the expression of migratory markers such as claudin-1 but a robust increase in E-cadherin expression in Smad4-SW480 clones compared with SW480 cells.
Re-establishment of Smad4 regulates the expression of E-cadherin and claudin-1 in SW480 cells
Smad4 restoration in SW480 cells decreases mRNA expression of bcatenin/Tcf target genes and inhibits cell migration
Many reports have demonstrated that E-cadherin and claudin-1 are also involved in the development of colon cancer. E-cadherin is known to negatively regulate b-catenin transcriptional activity of target genes through the recruitment of b-catenin from transcriptional complexes [12], claudin-1 is a known target molecule of the b-catenin/Tcf signaling pathway [10]. To determine whether Smad4 re-establishment has a causal role on E-cadherin and claudin-1 in tumor invasion and migration, we investigated the expression of E-cadherin and claudin-1 proteins by western blotting in Smad4-negative and Smad4-positive cells, respectively. As shown in Fig. 3A, low levels of E-cadherin protein were observed in SW480 cells and its control cell lines (pc-SW480) with relatively more E-cadherin detected in cells that had increased Smad4 (Smad4-SW480). Inverse data were found in cellular claudin-1 concentration, we observed reduced expression of claudin-1 in clones transfected with Smad4 when compared with the Smad4negative cells (Fig. 3A). We performed immunofluorescence analysis to further investigate whether up-regulation of Smad4
We further asked whether the decrease in abundance of b-catenin and its shift from nuclear/cytoplasmic location to the cell membrane regulated transcriptional activity of b-catenin/Tcf target genes, such as claudin-1 and MMP-7. The mRNA levels of claudin-1 and MMP-7 were examined by semiquantitative RT-PCR analysis as described in Methods. As shown in Fig. 4A, transfection of Smad4 resulted in reduced MMP-7 and claudin-1 mRNA concentration as compared to cells transfected with the control or nonsense. Many reports have documented that either claudin-1 over expression or loss of E-cadherin function has been implicated as mechanisms for cell invasion [1,13], and restoration of Smad4 by conventional means can inhibit cell invasion in many types of tumors [1]. The impact of increased Smad4 expression on colon cancer cells migration was evaluated using a boyden chamber migration assay. As shown in Fig. 4B, after cultivation of the cells for 24 h, migration of 117.8 ± 28.5 and 39.8 ± 12.1 cells per field of view through the porous transwells was observed for SW480 and Smad4-SW480, respectively. We observed a significant
X. Tian et al. / Biochemical and Biophysical Research Communications 380 (2009) 478–483
481
Fig. 2. Re-expression of Smad4 inhibits b-catenin/Tcf activity and induces relocalization of b-catenin from the cytoplasm and nucleus to the plasma membrane. (A) SW480, pc-SW480 and two Smad4-SW480 clones on 24-well plates were transiently transfected in triplicate with 0.8 lg of TOPflash or FOPflash reporter constructs, along with 0.016 lg of pRL-SV40 as internal control. Tcf-mediated gene transcription was determined by the ratio of pTOPflash to pFOPflash luciferase activity. Transfections were done in triplicate; columns, mean of three independent experiments; bars, SD. (B) In SW480 cells, b-catenin (green) was predominantly localized in cytoplasm and nucleus, whereas it was largely localized at cell membrane in Smad4-SW480 cells. Colocalization of b-catenin (green) with PI (red) is confirmed by overlay (yellow). (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
decrease in the migration capacity of Smad4 re-expression cells (Fig. 4B). Discussion Smad4 is the only co-Smad in mammalian cells and is potentially involved in signal transmission of all members of the TGFb superfamily. Recent studies disclosed that some of these TGFb pathways are, indeed, independent of Smad4. Wang et al confirmed that the TGFb induced adhesion response was independent of Smad4 while inactivation of Smad4 down-modulated the growth-inhibitory effect of TGFb in human colon carcinoma cells [14]. Muller et al. indicated that re-expression of Smad4 was not adequate to restore TGFb anti-proliferative responses in SW480 cells [8]. Here we showed a novel mechanism that underlies Smad4 function as a tumor suppressor. Our study confirmed that restoration of Smad4 in SW480 human colon carcinoma cells,
adequate to mediate tumor migration in vitro, was associated with the reduction of Wnt/b-catenin signaling, with decrease of b-catenin/Tcf target genes expression and with induction of functional Ecadherin expression. b-catenin in the nucleus is important for its cell proliferation related function [15]. In the experiments reported here, cytoplasmic and nuclear b-catenin protein levels were markedly reduced in Smad4-transfected cells, Smad4 up-regulation induced b-catenin translocation from the cytoplasm and nucleus to the cell membrane. These observations indicated that Smad4 was implicated in the modulation process of Wnt/b-catenin signaling, increased b-catenin degradation in cytoplasm and nucleus correlated with up-regulated Smad4 levels. It could be a direct interaction between Smad4 and b-catenin within the cytoplasm that promotes b-catenin degradation. Our data suggested that a potential mechanism of abnormal translocation of b-catenin to the membrane of colon cancer cells may be secondary to Smad4 over expression. We
482
X. Tian et al. / Biochemical and Biophysical Research Communications 380 (2009) 478–483
Fig. 3. Up-regulation of Smad4 results in increased E-cadherin and decreased claudin-1 expression. (A) Levels of E-cadherin and claudin-1 proteins were measured by immunoblot analysis using total protein from parental or control or 2 individual Smad4-SW480 clones as described in Methods. b-Actin was used as an internal control. (B) Localization of E-cadherin and claudin-1 was analyzed by indirect immunofluorescence and fluorescence microscope. In Smad4-SW480 cells, membranous staining of Ecadherin (green) was increased as compared to parental cells, and a visibly reduced staining of claudin-1 (red) in the cytoplasm was observed simultaneously. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
further asked whether the decrease in abundance of b-catenin and its shift of location was correlated with any decrease in Wnt signaling as reflected by b-catenin/Tcf transcriptional activity. Tcf/Lef luciferase reporter constructs is a well-established assay for measuring transcriptional activation of b-catenin/Tcf signaling [16]. By calculating the ratio of TOPflash to FOPflash luciferase activity in different cell lines, we confirmed that there was an inverse correlation between b-catenin/Tcf transcriptional activity and the expression of Smad4 in colon cancer cells. Restoration of Smad4 decreased relative luciferase activity in SW480 cells which suggests that Smad4 may have a direct inhibitory effect on b-catenin/Tcf signaling. It is thought that Smad4 expression depletes bcatenin from cytoplasm and nucleus, thus inhibiting Tcf/Lef activation. E-cadherin is a tumor suppressor protein with a well-established role in cell–cell adhesion [17]. Data from many studies suggest that loss of E-cadherin function elicits active signals that support tumor-cell migration and metastatic dissemination [18]. Although many different examples of signaling mediated by celladhesion molecules have been reported, the mechanism which Smsd4 expression regulates E-cadherin levels still remains to be elucidated. SW480 cells are characterized by very low level of Ecadherin expression, in the present study, we observed increased overall E-cadherin protein expression and fortified membranous
staining of E-cadherin in Smad4-transfected SW480 cells. Moreover, increased Smad4 in SW480 cells resulted in decreased migration as measured by a cell migration assay. E-cadherin is known to form a complex with b-catenin at adherens junctions, we have demonstrated the translocation of b-catenin to the cell membrane in Smad4-positive cells, therefore, increased membranous concentration of b-catenin enhanced the formation of the E-cadherin/bcatenin complex resulting in strengthening of cell–cell adhesion and decreased migration [18]. Anke Reinacher-Schick et al. have provided the evidence for a role of the tumor suppressor Smad4 as a positive regulator of the tumor and invasion suppressor E-cadherin [1], we further proved that E-cadherin expression was upregulated by Smad4 as a result of decreased expression of b-catenin in the cytoplasm and nucleus, relocalization of b-catenin to the plasma membrane. Previous experimental studies on claudin-1 and MMP-7 have indicated that the two genes are implicated in colon cancer migration and metastases. It has been reported that the levels of the two genes are positively correlated with the stage of CRC progression [19,20]. In this study, we observed the reduction in claudin-1 and MMP-7 mRNA expression in Smad4-expression cells. Both claudin1 and MMP-7 are known downstream target molecules of the Wnt/b-catenin signaling pathway, we considered that the reason of decreased mRNA expression was that Smad4 inhibited the b-cate-
X. Tian et al. / Biochemical and Biophysical Research Communications 380 (2009) 478–483
483
Fig. 4. Smad4 decreases the mRNA expression of b-catenin/Tcf target genes and migration activity of SW480 cells. (A) The mRNA expression of the b-catenin/Tcf target genes (claudin-1 and MMP-7) was determined by semiquantitative RT-PCR, results were normalized to GAPDH and presented as means ± SD of three independent experiments. There was less MMP-7 and claudin-1 mRNA in Smad4-SW480 clones than SW480 or pc-SW480 cells. (B) After cultivation of the three different cells for 24 h, the chamber migration assay was done. Migratory cell numbers were scored as described in Materials and methods. Columns, mean of three independent experiments done in triplicate; bars, SD. *p < 0.05, compared with SW480 cells. The p value was determined using Student’s t test.
nin/Tcf transcriptional activity. The following findings from immunoblot and immunofluorescence analysis also indicated that overexpression of Smad4 in SW480 cells induced a decrease in cytoplasmic claudin-1. These observations confirmed our hypothesis that a novel mechanism underlying the Smad4 migration-suppressive function through the regulation of the b-catenin/Tcf signal pathway. Taken together, results from this study demonstrated that Smad4 may play an important migration-suppressive role in colon carcinoma. This is probably mediated through suppressing Wnt/bcatenin transcriptional activity and relocating b-catenin from nucleus to the plasma membrane. Further understanding the functional mechanism of Smad4 as a tumor suppressor may provide a potential therapeutic strategy for intervention of colon cancer migration and metastasis.
[7]
[8]
[9]
[10]
[11]
[12]
Acknowledgments [13]
This study was supported by Institute for Digestive Medicine of Southern Medical University. The authors thank Dr. Xinying Wang for providing the TOPflash and FOPflash reporter plasmids.
[14]
References [15] [1] A. Reinacher-Schick, S.E. Baldus, B. Romdhana, et al., Loss of Smad4 correlates with loss of the invasion suppressor E-cadherin in advanced colorectal carcinomas, J. Pathol. 202 (2004) 412–420. [2] M. Miyaki, T. Kuroki, Role of Smad4 (DPC4) inactivation in human cancer, Biochem. Biophys. Res. Commun. 306 (2003) 799–804. [3] S. Chakrabarty, V. Radjendirane, H. Appelman, et al., Extracellular calcium and calcium sensing receptor function in human colon carcinomas: promotion of E-cadherin expression and suppression of beta-catenin/TCF activation, Cancer Res. 63 (2003) 67–71. [4] A. Gregorieff, H. Clevers, Wnt signaling in the intestinal epithelium: from endoderm to cancer, Genes Dev. 19 (2005) 877–890. [5] P. Polakis, The oncogenic activation of beta-catenin, Curr. Opin. Genet. Dev. 9 (1999) 15–21. [6] E. Labbe, A. Letamendia, L. Attisano, Association of Smads with lymphoid enhancer binding factor 1/T cell-specific factor mediates cooperative signaling
[16] [17]
[18] [19]
[20]
by the transforming growth factor-beta and wnt pathways, Proc. Natl. Acad. Sci. USA 97 (2000) 8358–8363. D.J. Satterwhite, K.L. Neufeld, TGF-beta targets the Wnt pathway components, APC and beta-catenin, as Mv1Lu cells undergo cell cycle arrest, Cell Cycle 3 (2004) 1069–1073. N. Muller, A. Reinacher-Schick, S. Baldus, et al., Smad4 induces the tumor suppressor E-cadherin and P-cadherin in colon carcinoma cells, Oncogene 21 (2002) 6049–6058. S.R. Shiou, A.B. Singh, K. Moorthy, et al., Smad4 regulates claudin-1 expression in a transforming growth factor-beta-independent manner in colon cancer cells, Cancer Res. 67 (2007) 1571–1579. N. Miwa, M. Furuse, S. Tsukita, et al., Involvement of claudin-1 in the betacatenin/Tcf signaling pathway and its frequent upregulation in human colorectal cancers, Oncol Res. 12 (2001) 469–476. K.L. Woodford-Richens, A.J. Rowan, P. Gorman, et al., SMAD4 mutations in colorectal cancer probably occur before chromosomal instability, but after divergence of the microsatellite instability pathway, Proc. Natl. Acad. Sci. USA 98 (2001) 9719–9723. A. Stockinger, A. Eger, J. Wolf, et al., E-cadherin regulates cell growth by modulating proliferation-dependent beta-catenin transcriptional activity, J. Cell Biol. 154 (2001) 1185–1196. N. Oku, E. Sasabe, E. Ueta, et al., Tight junction protein claudin-1 enhances the invasive activity of oral squamous cell carcinoma cells by promoting cleavage of laminin-5 gamma2 chain via matrix metalloproteinase (MMP)-2 and membrane-type MMP-1, Cancer Res. 66 (2006) 5251–5257. H. Wang, S. Rajan, G. Liu, et al., Transforming growth factor beta suppresses beta-catenin/Wnt signaling and stimulates an adhesion response in human colon carcinoma cells in a Smad4/DPC4 independent manner, Cancer Lett. 264 (2008) 281–287. M. Van de Wetering, E. Sancho, C. Verweij, et al., The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells, Cell 111 (2002) 241–250. K. Willert, R. Nusse, Beta-catenin: a key mediator of Wnt signaling, Curr. Opin. Genet. Dev. 8 (1998) 95–102. C.J. Gottardi, E. Wong, B.M. Gumbiner, E-cadherin suppresses cellular transformation by inhibiting beta-catenin signaling in an adhesionindependent manner, J. Cell Biol. 153 (2001) 1049–1060. U. Cavallaro, G. Christofori, Cell adhesion and signalling by cadherins and IgCAMs in cancer, Nat. Rev. Cancer 4 (2004) 118–132. Y. Adachi, H. Yamamoto, F. Itoh, et al., Contribution of matrilysin (MMP-7) to the metastatic pathway of human colorectal cancers, Gut 45 (1999) 252– 258. P. Dhawan, A.B. Singh, N.G. Deane, et al., Claudin-1 regulates cellular transformation and metastatic behavior in colon cancer, J. Clin. Invest. 115 (2005) 1765–1776.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Smad4 restoration leads to a suppression of Wnt/b-catenin signaling activity and migration capacity in human colon carcinoma cells Xiaoxiao Tian a, Hao Du b, Xiangsheng Fu a, Kang Li a, Aimin Li a, Yali Zhang a,* a b
Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou Road 1838, Guangzhou 510515, Guangdong Province, China Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, China
a r t i c l e
i n f o
Article history: Received 15 January 2009 Available online 25 January 2009
Keywords: Smad4 Tumor-suppressor gene Beta-catenin E-cadherin Colon cancer
a b s t r a c t Recent studies have reported that Smad4 has a TGF-b-independent function as a tumor suppressor in cooperating with b-catenin/Lef to regulate target gene expression. The objective of this research was to study the role of Smad4 in colon cancer migration and its potential mechanism. In our study, stable colon cancer cells that have increased Smad4 expression were created using a eukaryotic vector containing Smad4-pcDNA3.1(+). Smad4 restoration directly suppressed the Wnt/b-catenin signal activity in SW480 cells by down-regulating b-catenin expression and altering its localization from cytoplasm and nucleus to the plasma membrane. Up-regulation of Smad4 also increased the expression of E-cadherin and decreased the transcriptional activity of b-catenin/Tcf target genes, such as claudin-1 and MMP-7. SW480 cells with increased Smad4 expression had decreased in vitro cell migration. The data suggested that restoration of Smad4 in Smad4-deficient cells may provide a potential therapeutic strategy for intervention of colon cancer migration and metastasis. Ó 2009 Elsevier Inc. All rights reserved.
Smad4, originally characterized as a central intracellular signal transduction component of the transforming growth factor b (TGFb) family in epithelial cells, is regarded as a tumor suppressor gene predominantly involved in gastrointestinal carcinogenesis [1]. Loss of Smad4 function either due to loss of its expression or genetic mutation is considered to be a genetically late step and occurs in a significant proportion of colon and pancreatic cancer [2]. The Wnt/b-catenin signaling is implicated in colon carcinogenesis and suppression of this signal pathway is associated with a reduction of cellular proliferation and the induction of cellular differentiation [3]. b-catenin performs dual functions, which include a crucial role in cell–cell adhesion and the Wnt/b-catenin pathway [4]. Activation of Wnt signaling inhibits GSK dependent phosphorylation of b-catenin and results in an increase in b-catenin protein levels. The accumulation of b-catenin promotes its nuclear translocation where it regulates Wnt target genes transcription in collaboration with factors from the Tcf/Lef family [5]. Both TGFb and Wnt signaling pathways are thought to play critical roles in regulating the differentiation processes of colonic epithelial cells. Increasing evidence of cross talk between Smad signaling and the Wnt pathway has been reported during the past years [6,7]. Nevertheless, the relationship between the migrationsuppressive responses of Smad4 and the activity of Wnt signaling pathway is not thoroughly understood. Recent evidences have suggested additional mechanisms underlying the effects of Smad4, for * Corresponding author. Fax: +86 20 87280770. E-mail address: [email protected] (Y. Zhang). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.01.124
example, re-expression of Smad4 in the Smad4-deficient SW480 colon carcinoma cells resulted in loss of tumorigenicity of these cells in nude mice that was accompanied by the restoration of a more epithelioid morphology and induced transcription of E-cadherin and P-cadherin [8], in addition, it was recently shown that a novel mechanism underlying Smad4 invasion-suppressive function through regulation of a potential metastatic modulator, claudin-1, in a TGFb-independent manner [9]. Cell–cell adhesion in epithelial cell sheets is maintained mainly through adherens junctions and tight junctions [1]. E-cadherin is a key player in cell–cell adhesion connecting adjacent cells via the cadherin–catenin adhesion complex. Claudin-1, the most apical cell–cell contacts and the most important tight junction for barrier function in colon cancer, was recently identified as a target of Wnt/b-catenin signaling [10]. Based on these studies, we hypothesized that there was a relationship between Smad4, a tumor suppressor protein, and Wnt/b-catenin signaling, a progression-promoting pathway, in invasive and migratory colorectal carcinoma. The objective of this study was to investigate the impact of enhanced Smad4 expression on b-catenin and E-cadherin protein expression and localization in colon cancer cells and its effect on cell migration. In this study, we reported that Smad4 directly suppressed the Wnt/b-catenin signal pathway in Smad4-responsive human colon carcinoma cells by down-regulating the cytoplasmic and nucleus expression of b-catenin and increasing localization of b-catenin to the plasma membrane. We further showed that Smad4 induced the expression of the intercellular adhesion molecule E-cadherin and reduced transcriptional activity of b-catenin/
X. Tian et al. / Biochemical and Biophysical Research Communications 380 (2009) 478–483
Tcf target genes, such as claudin-1 and MMP-7. Lastly, we found that the above-mentioned reasons contributed to the migrationinhibitory effect of Smad4 in colon cancer cells. Materials and methods Cell cultures, plasmids and antibodies. Human colon carcinoma SW480 cells was obtained from ATCC and cultured in RPMI 1640 media supplemented with 10% fetal bovine serum. The full-length coding sequence of Smad4 was constructed into the eukaryotic expression vector pcDNA3.1(+) for stable transfection. The sequence of the recombinant plasmid was confirmed by sequencing. Empty vector pcDNA3.1(+) was used as control. The Lef/Tcf reporter plasmid, TOPflash, and its mutant control, FOPflash, were generously provided by Dr. Xinying Wang (Nanfang Hospital, Guangzhou, China). Primary immunoblotting antibodies were: anti-claudin-1 (Cell Signaling Technology, Beverly, MA), anti-Smad4, anti-b-catenin, anti-E-cadherin and anti-b-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA). All secondary antibodies used were obtained from Santa Cruz Biotechnology Inc. Overexpression of Smad4 by stable transfection. One day before transfection, the SW480 cells were seeded in 6-well cell culture plates to provide a final density of 60–70% confluence. Cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and selected in complete medium containing 1mg/ml G418 sulfate (Gibco BRL, USA) at 48h after transfection, G418-resistant cell clones were isolated for 2 weeks. After expansion of each individual clone, cells were screened for expression of Smad4 by immunoblot analysis. Two clones that expressed similar levels of Smad4 in protein concentrations were selected for subsequent experiments. SW480 cells that were stably transfected with pcDNA3.1(+) were used as a control. Immunoblot analysis. Cultured and harvested cells were lysed in RIPA buffer supplemented with protease inhibitors. Lysates were centrifuged at 15,000g for 30 min at 4 °C. To ascertain b-catenin cellular localization, cytoplasmic and nuclear proteins were isolated from cells using a cell fractionation kit (Keygen, NanJing, China). Equal amounts of protein samples were electrophoretically separated by SDS–PAGE in separation gels and transferred to nitrocellulose sheets. They were blocked in TBS-T containing 5% non-fat dry milk and then incubated overnight at 4 °C with the primary antibodies diluted in the same buffer (Smad4 1:400; E-cadherin 1:600; b-catenin 1:1000; claudin-1 1:1000; b-actin 1:3000 dilutions). Membranes were washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000 dilutions) for enhanced chemiluminescence detection using an ECL detection kit (Pierce Biotechnology Inc., Rockford, IL). Semiquantitative RT-PCR. Total RNA was isolated from the cell lines using the TRIzol reagent (Invitrogen, Carlsbad, CA). Reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit(Fermentas), 2 lg RNA as template and oligo(dT) as primer. PCR analysis was performed using gene-specific primers and diluted first-strand cDNA as template. Primers for Smad4: Forward 50 -GTGGCTGGTCGGAAAGGATT-30 , Reverse 50 -ACTGGCA GGCTGACTTGTGG-30 ; b-catenin: Forward 50 -ATCATCGTGAGGGCTT ACTG-30 , Reverse 50 -CCATCCCTTCCTGTTTAGTT-30 ; claudin-1: Forward 50 -GCCCTACTTTGCTGTTCCTG-30 ; Reverse 50 -CCCTTCCCCCAAT TGAGTAT-30 ; MMP-7: Forward 50 -TGGAGTGCCAGATGTTGCAG-30 , Reverse 50 -TTTCCATATAACTTCTGAATGCCT-30 ; GAPDH: Forward 50 -CGGGAAGCTTGTCATCAATGG-30 , Reverse 50 -GGCAGTGATGG CATGGACTG-30 . Immunofluorescence. Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% TritonX-100 solution and blocked in 3% bovine serum albumin–0.2% Triton X-100. Samples were then incubated with the following primary antibodies:
479
anti-b-catenin (mouse monoclonal); anti-claudin-1 and -E-cadherin (rabbit polyclonal). FITC anti-mouse and anti-rabbit, Rhodamine Red-X anti-rabbit were used as secondary antibodies at 1:200 dilutions, respectively. Stained glass coverslips were viewed and photographed using an OLYMPUS fluorescence microscope. Transient transfection and luciferase assays. For transient transfection assays, cells were seeded in 24-well plates in triplicate. Cells were cotransfected with a firefly luciferase reporter construct TOPflash or FOPflash, and a reference construct that contains Renilla reniformis luciferase, pRL-SV40 (Promega, Madison, WI), using Lipofectamine 2000. Forty-eight hours later, luciferase activities were measured using the Dual-Luciferase Reporter Assay System Kit (Promega) in a GloMax Luminometry System (Promega). Firefly luciferase activity was normalized to Renilla reniformis luciferase activity and plotted as mean ± SD from three independent experiments. Boyden chamber migration assay. Cells were resuspended in 0.2% bovine serum albumin serum-free medium at a concentration of 5 105 cells/ml, and 0.2 ml of each was seeded in the upper compartment of the chamber (24-well insert, 8 lm pore size; Millipore, Bedford, MA). About 0.6 ml appropriate medium supplemented with 10% fetal bovine serum was added to each well of the plate in the lower chamber. After 24 h of incubation, cells remaining inside the inserts were removed with cotton swabs, those that had migrated to the lower surface were fixed in formaldehyde and stained with crystal violet. Cells were counted in 5 random fields and expressed as the average number of cells per field under a light microscope. Each invasion assay was done in triplicate and repeated thrice. Results Smad4 restoration decreases b-catenin expression and alters its localization SW480 human colon carcinoma cells are Smad4-deficient, they lost one copy of chromosome 18, where Smad4 gene is located, the second Smad4 allele harbors a splice site mutation and is not expressed [11]. As shown in Fig. 1A and B, two clones of SW480 with stable expression of Smad4 were established using Smad4 eukaryotic expression plasmid. This cell line was established so that the effects of Smad4 re-expression on Wnt signaling activity could be compared. To determine whether Smad4 regulates b-catenin protein or mRNA expression, we prepared total RNA and cytoplasmic and nuclear proteins from parental, pc-SW480 (expressing empty vector) and Smad4-SW480 (overexpressing Smad4) cells, respectively, then subjected them to semiquantitative RT-PCR and immunoblot analysis as described in Methods. We detected a 40% reduction in the levels of cytoplasmic b-catenin and 60% in the nucleus in Smad4-SW480 clones when compared with SW480 cells (Fig. 1B). A 40% reduction in b-catenin mRNA expression was observed in Smad4-transfected cells (Fig. 1A). As the function of bcatenin in signal transduction depends on its cytoplasmic/nuclear localization, we further analyzed the distribution of the b-catenin protein through indirect immunofluorescence. SW480 cells showed strong staining for b-catenin in the cytoplasm and nucleus. Smad4 re-expression cells, in contrast, showed staining in the plasma membrane. The data demonstrated that up-regulation of Smad4 caused the relocalization of b-catenin from cytoplasm and nucleus to the plasma membrane in SW480 cells (Fig. 2B). Re-expression of Smad4 in SW480 cells leads to a reduction in bcatenin/Tcf signaling The presence of low cytoplasmic and nuclear levels of b-catenin suggested that Wnt/b-catenin signaling was impaired in Smad4SW480 cells. To explore the possibility, we analyzed b-catenin/
480
X. Tian et al. / Biochemical and Biophysical Research Communications 380 (2009) 478–483
Fig. 1. SW480 cells with increased Smad4 expression and its effect on b-catenin expression. Cells were transfected with recombinant plasmid Smad4-pcDNA3.1(+) and two Smad4-SW480 clones were obtained. (A) Total RNA from the indicated cells were isolated as described in Methods and subjected to RT-PCR for Smad4 and b-catenin. The results were normalized to GAPDH and presented as means ± SD of three independent experiments. There was less b-catenin mRNA expression in Smad4-transfected cells than Smad4-negative colon carcinoma cells (p < 0.05). (B) Immunoblot analysis for the expression of Smad4 and b-catenin in SW480, pc-SW480 and two Smad4-SW480 clones, b-actin was used as a control for protein loading. The E-cadherin protein expression levels were normalized to b-actin and the results were presented as means ± SD of three independent experiments. Relatively less b-catenin was detected in the cytoplasmic and nuclear fractions of Smad4-transfected cells compared with SW480 and pcSW480 groups.
Tcf signaling activity in SW480, pc-SW480 and Smad4-SW480 cells by transiently transfecting TCF and mutant TCF reporter plasmids (TOPflash and FOPflash, respectively). To control for transfection efficiency, we cotransfected a pRL-SV40 reporter construct as internal control. Relative luciferase activities were calculated as described in Methods. As shown in Fig. 2A, the relative transcriptional activity of the b-catenin/Lef complex was decreased (3- to 4-fold) in Smad4-SW480 cells compared with SW480 or pcSW480 cells. This analysis confirmed that Wnt signaling activity was significantly suppressed by Smad4 expression.
expression changed the localization of E-cadherin and claudin-1. The localization of E-cadherin and claudin-1 was not altered in Smad4-SW480 cells, however, restoration of Smad4 resulted in increased E-cadherin expression at the cell membrane and reduced claudin-1 expression in the cytoplasm (Fig. 3B). The observations were coincident with immunoblot analysis of the two proteins. The results of these two studies indicated a decrease in the expression of migratory markers such as claudin-1 but a robust increase in E-cadherin expression in Smad4-SW480 clones compared with SW480 cells.
Re-establishment of Smad4 regulates the expression of E-cadherin and claudin-1 in SW480 cells
Smad4 restoration in SW480 cells decreases mRNA expression of bcatenin/Tcf target genes and inhibits cell migration
Many reports have demonstrated that E-cadherin and claudin-1 are also involved in the development of colon cancer. E-cadherin is known to negatively regulate b-catenin transcriptional activity of target genes through the recruitment of b-catenin from transcriptional complexes [12], claudin-1 is a known target molecule of the b-catenin/Tcf signaling pathway [10]. To determine whether Smad4 re-establishment has a causal role on E-cadherin and claudin-1 in tumor invasion and migration, we investigated the expression of E-cadherin and claudin-1 proteins by western blotting in Smad4-negative and Smad4-positive cells, respectively. As shown in Fig. 3A, low levels of E-cadherin protein were observed in SW480 cells and its control cell lines (pc-SW480) with relatively more E-cadherin detected in cells that had increased Smad4 (Smad4-SW480). Inverse data were found in cellular claudin-1 concentration, we observed reduced expression of claudin-1 in clones transfected with Smad4 when compared with the Smad4negative cells (Fig. 3A). We performed immunofluorescence analysis to further investigate whether up-regulation of Smad4
We further asked whether the decrease in abundance of b-catenin and its shift from nuclear/cytoplasmic location to the cell membrane regulated transcriptional activity of b-catenin/Tcf target genes, such as claudin-1 and MMP-7. The mRNA levels of claudin-1 and MMP-7 were examined by semiquantitative RT-PCR analysis as described in Methods. As shown in Fig. 4A, transfection of Smad4 resulted in reduced MMP-7 and claudin-1 mRNA concentration as compared to cells transfected with the control or nonsense. Many reports have documented that either claudin-1 over expression or loss of E-cadherin function has been implicated as mechanisms for cell invasion [1,13], and restoration of Smad4 by conventional means can inhibit cell invasion in many types of tumors [1]. The impact of increased Smad4 expression on colon cancer cells migration was evaluated using a boyden chamber migration assay. As shown in Fig. 4B, after cultivation of the cells for 24 h, migration of 117.8 ± 28.5 and 39.8 ± 12.1 cells per field of view through the porous transwells was observed for SW480 and Smad4-SW480, respectively. We observed a significant
X. Tian et al. / Biochemical and Biophysical Research Communications 380 (2009) 478–483
481
Fig. 2. Re-expression of Smad4 inhibits b-catenin/Tcf activity and induces relocalization of b-catenin from the cytoplasm and nucleus to the plasma membrane. (A) SW480, pc-SW480 and two Smad4-SW480 clones on 24-well plates were transiently transfected in triplicate with 0.8 lg of TOPflash or FOPflash reporter constructs, along with 0.016 lg of pRL-SV40 as internal control. Tcf-mediated gene transcription was determined by the ratio of pTOPflash to pFOPflash luciferase activity. Transfections were done in triplicate; columns, mean of three independent experiments; bars, SD. (B) In SW480 cells, b-catenin (green) was predominantly localized in cytoplasm and nucleus, whereas it was largely localized at cell membrane in Smad4-SW480 cells. Colocalization of b-catenin (green) with PI (red) is confirmed by overlay (yellow). (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
decrease in the migration capacity of Smad4 re-expression cells (Fig. 4B). Discussion Smad4 is the only co-Smad in mammalian cells and is potentially involved in signal transmission of all members of the TGFb superfamily. Recent studies disclosed that some of these TGFb pathways are, indeed, independent of Smad4. Wang et al confirmed that the TGFb induced adhesion response was independent of Smad4 while inactivation of Smad4 down-modulated the growth-inhibitory effect of TGFb in human colon carcinoma cells [14]. Muller et al. indicated that re-expression of Smad4 was not adequate to restore TGFb anti-proliferative responses in SW480 cells [8]. Here we showed a novel mechanism that underlies Smad4 function as a tumor suppressor. Our study confirmed that restoration of Smad4 in SW480 human colon carcinoma cells,
adequate to mediate tumor migration in vitro, was associated with the reduction of Wnt/b-catenin signaling, with decrease of b-catenin/Tcf target genes expression and with induction of functional Ecadherin expression. b-catenin in the nucleus is important for its cell proliferation related function [15]. In the experiments reported here, cytoplasmic and nuclear b-catenin protein levels were markedly reduced in Smad4-transfected cells, Smad4 up-regulation induced b-catenin translocation from the cytoplasm and nucleus to the cell membrane. These observations indicated that Smad4 was implicated in the modulation process of Wnt/b-catenin signaling, increased b-catenin degradation in cytoplasm and nucleus correlated with up-regulated Smad4 levels. It could be a direct interaction between Smad4 and b-catenin within the cytoplasm that promotes b-catenin degradation. Our data suggested that a potential mechanism of abnormal translocation of b-catenin to the membrane of colon cancer cells may be secondary to Smad4 over expression. We
482
X. Tian et al. / Biochemical and Biophysical Research Communications 380 (2009) 478–483
Fig. 3. Up-regulation of Smad4 results in increased E-cadherin and decreased claudin-1 expression. (A) Levels of E-cadherin and claudin-1 proteins were measured by immunoblot analysis using total protein from parental or control or 2 individual Smad4-SW480 clones as described in Methods. b-Actin was used as an internal control. (B) Localization of E-cadherin and claudin-1 was analyzed by indirect immunofluorescence and fluorescence microscope. In Smad4-SW480 cells, membranous staining of Ecadherin (green) was increased as compared to parental cells, and a visibly reduced staining of claudin-1 (red) in the cytoplasm was observed simultaneously. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
further asked whether the decrease in abundance of b-catenin and its shift of location was correlated with any decrease in Wnt signaling as reflected by b-catenin/Tcf transcriptional activity. Tcf/Lef luciferase reporter constructs is a well-established assay for measuring transcriptional activation of b-catenin/Tcf signaling [16]. By calculating the ratio of TOPflash to FOPflash luciferase activity in different cell lines, we confirmed that there was an inverse correlation between b-catenin/Tcf transcriptional activity and the expression of Smad4 in colon cancer cells. Restoration of Smad4 decreased relative luciferase activity in SW480 cells which suggests that Smad4 may have a direct inhibitory effect on b-catenin/Tcf signaling. It is thought that Smad4 expression depletes bcatenin from cytoplasm and nucleus, thus inhibiting Tcf/Lef activation. E-cadherin is a tumor suppressor protein with a well-established role in cell–cell adhesion [17]. Data from many studies suggest that loss of E-cadherin function elicits active signals that support tumor-cell migration and metastatic dissemination [18]. Although many different examples of signaling mediated by celladhesion molecules have been reported, the mechanism which Smsd4 expression regulates E-cadherin levels still remains to be elucidated. SW480 cells are characterized by very low level of Ecadherin expression, in the present study, we observed increased overall E-cadherin protein expression and fortified membranous
staining of E-cadherin in Smad4-transfected SW480 cells. Moreover, increased Smad4 in SW480 cells resulted in decreased migration as measured by a cell migration assay. E-cadherin is known to form a complex with b-catenin at adherens junctions, we have demonstrated the translocation of b-catenin to the cell membrane in Smad4-positive cells, therefore, increased membranous concentration of b-catenin enhanced the formation of the E-cadherin/bcatenin complex resulting in strengthening of cell–cell adhesion and decreased migration [18]. Anke Reinacher-Schick et al. have provided the evidence for a role of the tumor suppressor Smad4 as a positive regulator of the tumor and invasion suppressor E-cadherin [1], we further proved that E-cadherin expression was upregulated by Smad4 as a result of decreased expression of b-catenin in the cytoplasm and nucleus, relocalization of b-catenin to the plasma membrane. Previous experimental studies on claudin-1 and MMP-7 have indicated that the two genes are implicated in colon cancer migration and metastases. It has been reported that the levels of the two genes are positively correlated with the stage of CRC progression [19,20]. In this study, we observed the reduction in claudin-1 and MMP-7 mRNA expression in Smad4-expression cells. Both claudin1 and MMP-7 are known downstream target molecules of the Wnt/b-catenin signaling pathway, we considered that the reason of decreased mRNA expression was that Smad4 inhibited the b-cate-
X. Tian et al. / Biochemical and Biophysical Research Communications 380 (2009) 478–483
483
Fig. 4. Smad4 decreases the mRNA expression of b-catenin/Tcf target genes and migration activity of SW480 cells. (A) The mRNA expression of the b-catenin/Tcf target genes (claudin-1 and MMP-7) was determined by semiquantitative RT-PCR, results were normalized to GAPDH and presented as means ± SD of three independent experiments. There was less MMP-7 and claudin-1 mRNA in Smad4-SW480 clones than SW480 or pc-SW480 cells. (B) After cultivation of the three different cells for 24 h, the chamber migration assay was done. Migratory cell numbers were scored as described in Materials and methods. Columns, mean of three independent experiments done in triplicate; bars, SD. *p < 0.05, compared with SW480 cells. The p value was determined using Student’s t test.
nin/Tcf transcriptional activity. The following findings from immunoblot and immunofluorescence analysis also indicated that overexpression of Smad4 in SW480 cells induced a decrease in cytoplasmic claudin-1. These observations confirmed our hypothesis that a novel mechanism underlying the Smad4 migration-suppressive function through the regulation of the b-catenin/Tcf signal pathway. Taken together, results from this study demonstrated that Smad4 may play an important migration-suppressive role in colon carcinoma. This is probably mediated through suppressing Wnt/bcatenin transcriptional activity and relocating b-catenin from nucleus to the plasma membrane. Further understanding the functional mechanism of Smad4 as a tumor suppressor may provide a potential therapeutic strategy for intervention of colon cancer migration and metastasis.
[7]
[8]
[9]
[10]
[11]
[12]
Acknowledgments [13]
This study was supported by Institute for Digestive Medicine of Southern Medical University. The authors thank Dr. Xinying Wang for providing the TOPflash and FOPflash reporter plasmids.
[14]
References [15] [1] A. Reinacher-Schick, S.E. Baldus, B. Romdhana, et al., Loss of Smad4 correlates with loss of the invasion suppressor E-cadherin in advanced colorectal carcinomas, J. Pathol. 202 (2004) 412–420. [2] M. Miyaki, T. Kuroki, Role of Smad4 (DPC4) inactivation in human cancer, Biochem. Biophys. Res. Commun. 306 (2003) 799–804. [3] S. Chakrabarty, V. Radjendirane, H. Appelman, et al., Extracellular calcium and calcium sensing receptor function in human colon carcinomas: promotion of E-cadherin expression and suppression of beta-catenin/TCF activation, Cancer Res. 63 (2003) 67–71. [4] A. Gregorieff, H. Clevers, Wnt signaling in the intestinal epithelium: from endoderm to cancer, Genes Dev. 19 (2005) 877–890. [5] P. Polakis, The oncogenic activation of beta-catenin, Curr. Opin. Genet. Dev. 9 (1999) 15–21. [6] E. Labbe, A. Letamendia, L. Attisano, Association of Smads with lymphoid enhancer binding factor 1/T cell-specific factor mediates cooperative signaling
[16] [17]
[18] [19]
[20]
by the transforming growth factor-beta and wnt pathways, Proc. Natl. Acad. Sci. USA 97 (2000) 8358–8363. D.J. Satterwhite, K.L. Neufeld, TGF-beta targets the Wnt pathway components, APC and beta-catenin, as Mv1Lu cells undergo cell cycle arrest, Cell Cycle 3 (2004) 1069–1073. N. Muller, A. Reinacher-Schick, S. Baldus, et al., Smad4 induces the tumor suppressor E-cadherin and P-cadherin in colon carcinoma cells, Oncogene 21 (2002) 6049–6058. S.R. Shiou, A.B. Singh, K. Moorthy, et al., Smad4 regulates claudin-1 expression in a transforming growth factor-beta-independent manner in colon cancer cells, Cancer Res. 67 (2007) 1571–1579. N. Miwa, M. Furuse, S. Tsukita, et al., Involvement of claudin-1 in the betacatenin/Tcf signaling pathway and its frequent upregulation in human colorectal cancers, Oncol Res. 12 (2001) 469–476. K.L. Woodford-Richens, A.J. Rowan, P. Gorman, et al., SMAD4 mutations in colorectal cancer probably occur before chromosomal instability, but after divergence of the microsatellite instability pathway, Proc. Natl. Acad. Sci. USA 98 (2001) 9719–9723. A. Stockinger, A. Eger, J. Wolf, et al., E-cadherin regulates cell growth by modulating proliferation-dependent beta-catenin transcriptional activity, J. Cell Biol. 154 (2001) 1185–1196. N. Oku, E. Sasabe, E. Ueta, et al., Tight junction protein claudin-1 enhances the invasive activity of oral squamous cell carcinoma cells by promoting cleavage of laminin-5 gamma2 chain via matrix metalloproteinase (MMP)-2 and membrane-type MMP-1, Cancer Res. 66 (2006) 5251–5257. H. Wang, S. Rajan, G. Liu, et al., Transforming growth factor beta suppresses beta-catenin/Wnt signaling and stimulates an adhesion response in human colon carcinoma cells in a Smad4/DPC4 independent manner, Cancer Lett. 264 (2008) 281–287. M. Van de Wetering, E. Sancho, C. Verweij, et al., The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells, Cell 111 (2002) 241–250. K. Willert, R. Nusse, Beta-catenin: a key mediator of Wnt signaling, Curr. Opin. Genet. Dev. 8 (1998) 95–102. C.J. Gottardi, E. Wong, B.M. Gumbiner, E-cadherin suppresses cellular transformation by inhibiting beta-catenin signaling in an adhesionindependent manner, J. Cell Biol. 153 (2001) 1049–1060. U. Cavallaro, G. Christofori, Cell adhesion and signalling by cadherins and IgCAMs in cancer, Nat. Rev. Cancer 4 (2004) 118–132. Y. Adachi, H. Yamamoto, F. Itoh, et al., Contribution of matrilysin (MMP-7) to the metastatic pathway of human colorectal cancers, Gut 45 (1999) 252– 258. P. Dhawan, A.B. Singh, N.G. Deane, et al., Claudin-1 regulates cellular transformation and metastatic behavior in colon cancer, J. Clin. Invest. 115 (2005) 1765–1776.