Research Article 2053 CTGF enhances the motility of breast cancer cells via an integrin-␣v3–ERK1/2-dependent S100A4- upregulated pathway
Pai-Sheng Chen1,2,*, Ming-Yang Wang1,2,3,*, Shin-Ni Wu1, Jen-Liang Su4,5,6, Chih-Chen Hong7, Shuang-En Chuang7, Min-Wei Chen1, Kuo-Tai Hua1, Yu-Ling Wu1, Shih-Ting Cha1, Munisamy Suresh Babu1, Chiung-Nien Chen2,3, Po-Huang Lee2,3, King-Jen Chang2,3,‡,§ and Min-Liang Kuo1,2,‡,§ 1Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology, College of Medicine and 2Angiogenesis Research Center, National Taiwan University, Taipei, Taiwan 3Department of Surgery, National Taiwan University Hospital, Taipei 100, Taiwan 4Graduate Institute of Cancer Biology, College of Medicine, China Medical University, Taichung 404, Taiwan 5Center for Molecular Medical, China Medical University Hospital, Taichung 404, Taiwan 6Department of Biotechnology and Bioinformatics, Asia University, Taichung 41354, Taiwan 7Division of Cancer Research, National Health Research Institutes, Taipei 10016, Taiwan *These authors contributed equally to this work ‡Authors for correspondence (e-mails: [email protected]; [email protected]) §These authors contributed equally to this work
Accepted 11 April 2007 Journal of Cell Science 120, 2053-2065 Published by The Company of Biologists 2007 doi:10.1242/jcs.03460
Summary Connective tissue growth factor (CTGF) expression is S100A4 reversed the CTGF-induced cellular migratory elevated in advanced stages of breast cancer, but the ability, whereas overexpression of S100A4 in MDA231/AS regulatory role of CTGF in invasive breast cancer cell cells restored their high migratory ability. Genetic and phenotypes is unclear. Presently, overexpression of CTGF pharmacological manipulations suggested that the CTGF- in MCF-7 cells (MCF-7/CTGF cells) enhanced cellular mediated S100A4 upregulation was dependent on ERK1/2 migratory ability and spindle-like morphological activation, with expression levels of CTGF and S100A4 alterations, as evidenced by actin polymerization and focal- being closely correlated with human breast tumors. We adhesion-complex aggregation. Reducing the CTGF level conclude that CTGF plays a crucial role in in MDA-MB-231 (MDA231) cells by antisense CTGF cDNA migratory/invasive processes in human breast cancer by a (MDA231/AS cells) impaired cellular migration and mechanism involving activation of the integrin- ␣  Journal of Cell Science promoted a change to an epithelial-like morphology. A v 3–ERK1/2–S100A4 pathway. neutralizing antibody against integrin ␣v3 significantly attenuated CTGF-mediated ERK1/2 activation and cellular migration, indicating that the integrin- Supplementary material available online at ␣v3–ERK1/2 signaling pathway is crucial in mediating http://jcs.biologists.org/cgi/content/full/120/12/2053/DC1 CTGF function. Moreover, the cDNA microarray analysis revealed CTGF-mediated regulation of the prometastatic Key words: Connective tissue growth factor, Breast cancer, ERK1/2, gene S100A4. Transfection of MCF-7/CTGF cells with AS- Integrin, S100A4, Cell migration, F-actin
Introduction derived factor 1 (CXCL12) confer ameliorated migratory Metastasis, the major cause of mortality for cancer patients, is ability to breast cancer cells and are correlated with more a complex and multi-stage process in which secondary tumors advanced-stages of breast cancer (Darash-Yahana et al., 2004; are formed in distant sites (Van’t Veer and Weigelt, 2003). Miralem et al., 2001; Price et al., 1999; Tangkeangsirisin and Typically, the development of metastasis involves several steps Serrero, 2004). These molecules have been proposed as that comprise cellular transformation and tumor growth, therapeutic targets (Barrett-Lee, 2005). Identification and angiogenesis and lymphangiogenesis, entry of cancer cells into molecular characterization of new molecules that are involved the circulation by intravasation, anchorage and/or attachment in tumor progression, therefore, have important clinical in the target organ, invasion of the target organ by implications. extravasation, and proliferation within the organ parenchyma In order to invade, a tumor cell must undergo major changes (Hanahan and Weinberg, 2000). The migratory ability of a in shape. Cellular motility depends on localized actin cancer cell is important for many of these steps, and therefore polymerization at the leading edge of the cells (Kirfel et al., is correlated with tumor metastasis. In particular, several 2004), and the polymerization and depolymerization of actin secreted proteins, including vascular endothelial growth factor filaments must be under dynamic control. Simultaneously, (VEGF), PC-cell-derived growth factor (PCDGF/GP88, paxillin and vinculin interact at focal contacts of the actin stress progranulin), epidermal growth factor (EGF), and stromal-cell- fibers, providing a link to the extracellular matrix (e.g. 2054 Journal of Cell Science 120 (12)
fibronectin and vitronectin). These cytoskeletal changes enable MDA231 and MDA-MB-435. The breast cancer cell lines the invading cell to pass through the stromal cells, extracellular BT474, BT483, T47D, MDA-MB-453 and MCF-7 expressed matrix and endothelial cell layer. Integrins, paxillin, selectins, extremely low or undetectable levels of CTGF mRNA, transmembrane receptor tyrosine kinases, phospholipids, focal whereas expression was high in MDA231 and MDA-MB-435 adhesion kinases (FAKs), GTPases and the S100 calcium (Fig. 1A). No correlation was found between CTGF binding protein A4 (S100A4) calcium binding protein have expression and their histological types. Since cell migration is been described as being involved in regulating the organization crucial for cancer cell invasiveness, we next tested the cellular of the actin cytoskeleton (Kim and Helfman, 2003; Turner, migratory ability by a wound healing migration assay. 2000; Brunton et al., 2004). Some of these molecules have also MDA231 and MDA-MB-435 cells that exuberantly expressed been implicated in the malignant phenotype of certain CTGF mRNA exhibited a higher level of migratory ability carcinomas, such as ERBB-2, in mammary carcinomas than the cell lines with a lower level of CTGF mRNA (Fig. (Mariani et al., 2005). ERBB2 increases the potential for 1B). To assess whether CTGF was directly involved in the metastasis by upregulating the expression of the prometastatic regulation of breast cancer cell motility, both non-migrating S100A4 in medulloblastoma (Hernan et al., 2003). MCF-7 and migrating MDA231 cells were employed to Connective tissue growth factor (CTGF, also known as generate the CTGF-overexpression (MCF-7/CTGF) and CCN2) belongs to the CCN family (Bork, 1993). This family antisense-CTGF expression (MDA231/AS) cells, respectively. consists of six members, CTGF, NOVH, CYR61, WISP1, The CTGF expression levels in vector control cells and stable WISP2 and WISP3 (Perbal, 2004) that all possess an N- transfectants was compared using RT-PCR and western terminal signal peptide identifying them as secreted proteins. blotting. Both mRNA and protein levels of CTGF were CCN proteins probably carry out their biological activity significantly higher in MCF-7/CTGF than in MCF-7/neo cells through binding and activating of the cell surface integrins, (Fig. 1C). However, expression of CTGF was dramatically accompanied by activation of Akt and/or MAPK signal inhibited by antisense-CTGF orientation in MDA231/AS cascades (Perbal, 2004). The biological properties of CCN cells. Since, CTGF has already been reported to act as a proteins involve the stimulation of cellular proliferation, mitogen in endothelial cells (Shimo et al., 1999), we sought migration, adhesion, extracellular matrix formation, and also to characterize the cellular growth rate of control cells and the regulation of angiogenesis and tumorigenesis (Lau and transfectants, by performing the MTT assay 1-6 days after cell Lam, 1999). Overexpression of CTGF, WISP1, and CYR61 in seeding (Fig. 1D). No appreciable difference in cell growth breast tumor cells have been linked to tumor size and lymph ability was evident among these cells (Fig. 1D), suggesting node metastasis (Xie et al., 2001), suggesting that these CCN that CTGF does not have any mitogenic effect in human breast proteins are involved in the progression of breast cancer. cancer cells. Furthermore, the migratory ability of these Recently, we reported that CYR61 influences the resistance to transfectants was analyzed using a Boyden-chamber migration chemotherapeutic-agent-induced apoptosis in human breast assay. As shown in Fig. 1E, the overexpression of CTGF cancer MCF-7 cells (Lin et al., 2004). However, the biological increased the migratory ability by approximately 1.7-fold in activities of CTGF in breast cancer have not yet been explored. MCF-7 cells. Knockdown of CTGF expression inhibited the
Journal of Cell Science CTGF serves as an angiogenic factor in collaboration with migratory ability by approximately 60% in MDA231 cells matrix metalloproteinases (Kondo et al., 2002). Use of in vitro (Fig. 1F). and in vivo selection models and large-scale microarray analysis has revealed that CTGF crucial for the formation of CTGF promotes the dynamic regulation of actin osteolytic bone metastasis in breast cancer (Kang et al., 2003; structures and the formation of focal contact sites in Minn et al., 2005). Thus, CTGF plays an important role in breast cancer cells breast cancer progression. However, the precise role of CTGF Transfection of MCF-7 cells with CTGF led to a change in in breast cancer metastasis is still unknown. cellular morphology (Fig. 2a,e). MCF-7/CTGF cells became In this study, we demonstrate that CTGF can modulate the more spindle-like with long, protruding filamentous processes. cytoskeletal reorganization and in vitro migratory behavior of Double staining of the MCF-7/neo cells using antibodies breast cancer cells. We show that activation of ERK1/2 through conjugated to Texas-Red-conjugated phalloidin and integrin ␣v3 confers the enhanced cellular motility. monoclonal anti-paxillin (the latter as a marker for focal Microarray and reverse transcription (RT)-PCR analysis adhesions) revealed few, if any, actin stress fibers, whereas revealed that the crucial prometastatic S100A4 is significantly paxillin was diffusely distributed in the cells (Fig. 2b,c,d). The upregulated in cells and breast tumors that overexpress CTGF. MCF-7/CTGF cells exhibited numerous stress fibers (Fig. 2f) Our results support a new mechanism, in which integrin- with patches of paxillin staining appearing at the leading edges ␣v3–ERK1/2-dependent upregulation of S100A4 contributes of actin stress fibers (Fig. 2f,g,h). By contrast, overexpression to CTGF-enhanced migratory ability. of CTGF changed the MDA231 cell morphology from spindle- like to epithelial-like (Fig. 2i,m). In MDA231/neo cells, a Results plethora of both actin stress fibers and paxillin were evident, CTGF enhances the migratory ability of human breast and paxillin was macroaggregated at focal adhesions connected cancer cells to actin stress fibers (Fig. 2i,j,k). Reducing the level of CTGF levels are correlated with most of the advanced stages endogenous CTGF by transfection with CTGF/AS of breast cancers (Xie et al., 2001). Hence, we investigated significantly decreased the amount of actin stress fibers and expression levels of CTGF by northern blot analysis in several paxillin (Fig. 2n,o,p). These results support the hypothesis that adeno/ductal carcinoma of human breast cancer cell lines CTGF promotes actin contractility events and the dynamic including BT474, BT483, T47D, MDA-MB-453, MCF-7, regulation of focal contact structures in breast cancer cells. CTGF promotes breast cancer metastasis 2055
Fig. 1. CTGF expression enhances migratory ability in human breast cancer cells. (A) Expression of CTGF was detected in BT474, BT483,
Journal of Cell Science T47D, MDA-MB-453, MCF-7, MDA-MB-231, and MDA-MB-435 cells using northern blotting. (B) The migratory ability of the breast cancer cells was tested using a wound healing migration assay. Cells were seeded at confluence under normal culture conditions for 24 hours. Monolayers were wounded by scratching with a pipette tip. Images were taken at 20ϫ magnification. Three wells per experiment were counted and each experiment was repeated three times, error bars are the corresponding upper 95% confidence intervals. (C) CTGF expression levels in transfected MCF-7 and MDA-MB-231 cells. Expression of CTGF was detected in MCF-7/neo, MCF-7/CTGF, MDA231/neo, and MDA231/AS cells by RT-PCR (upper panels) and western blotting (lower panels). (D) Effects of CTGF on cellular growth. Cells were seeded on 24-well dishes and cell growth was assayed using a MTT assay. (E,F) Effects of CTGF on the migratory abilities of MCF-7/neo, MCF-7/CTGF, MDA231/neo, and MDA231/AS cells. The migratory ability was measured by using a Boyden chamber assay. Each of the transfected cells was tested in three separate experiments with incubations conducted in triplicate. Columns show the means of three independent experiments, and the error bars are the corresponding upper 95% confidence intervals. Asterisks denote a statistically significant difference in migratory ability of cells transfected with sense- or antisense-CTGF compared to cells transfected with the empty vector (*P<0.05, **P<0.01, two-tailed Student’s t-test).
The CTGF–integrin-␣v3 axis contributes to the changed from a spindle-like to an epithelial-like morphology enhancement of cellular migration and morphological by treatment with CTGF-neutralizing antibody in MDA231 changes cells (Fig. 3Da,e,i). Immunofluorescence staining showed that Since CTGF is a secreted protein, we explored the mechanism both actin stress fibers and paxillin-containing focal adhesions by which CTGF-mediated outside-in signals involved in were disrupted in CTGF-neutralizing antibody-treated CTGF-induced cellular migration using purified Fc-tagged MDA231 cells (Fig. 3D). recombinant CTGF (rCTGF) and the conditioned medium Integrins ␣v3, ␣IIb3, ␣M2 and ␣51 are classical cell- (CM) derived from MDA231 (MDA231/CM) to treat MCF-7 surface receptors of CTGF (Babic et al., 1999; Jedsadayanmata cells. Both rCTGF and MDA231/CM effectively enhanced the et al., 1999; Schober et al., 2002; Weston et al., 2003). Among MCF-7 cell migratory ability (Fig. 3A,B). CTGF-neutralizing these integrins, ␣v3 is the only one that associates with the antibody reversed the MDA231/CM-induced migration in a invasiveness in human breast cell lines and advanced tumor dose-dependent manner (Fig. 3B). Similar effects were also progression (Berry et al., 2004; Damjanovich et al., 1997; found in wild-type MDA231 cells, and the migratory ability of Felding-Habermann et al., 2001; Gui et al., 1996; Jones et al., MDA231 was dose dependently inhibited by treatment with 1995). Treatment with integrin-␣v3-blocking antibody dose CTGF-neutralizing antibody (Fig. 3C). Simultaneously, cells dependently reversed the CTGF-induced migratory ability 2056 Journal of Cell Science 120 (12)
(Fig. 3E), suggesting that integrin ␣v3 is a key receptor for CTGF-enhanced migratory ability was reduced when CTGF- CTGF-mediated signaling pathway. Moreover, CTGF mediated ERK1/2 phosphorylation was inhibited by PD98059 expression could upregulate integrin 3 expression and inhibitor in MCF-7/CTGF cells (Fig. 4B). However, integrin ␣v3 formation (supplementary material Fig. S1), transfection with active mutant MEK1 caused ERK1/2 re- suggesting the amplification loop strengthens the CTGF-␣v3 phosphorylation in MDA231/AS cells, leading to the signaling pathway. Taken together, our results indicate that enhancement of migratory ability (Fig. 4C). These results CTGF promotes cytoskeleton rearrangement and migratory indicated that the ERK1/2 pathway is essential for CTGF- ability by an autocrine/paracrine outside-in signaling manner mediated cell migration. through binding to integrin ␣v3 in human breast cancer cells. The recombinant CTGF protein also dramatically induced ERK1/2 phosphorylation within 10-60 minutes (Fig. 4D). CTGF activation of the ERK1/2 pathway via integrin Treatment with integrin-␣v3-blocking antibody inhibited the ␣v3 is required for cell motility rCTGF-induced ERK1/2 phosphorylation in MCF-7 cells (Fig. To investigate the possible signaling pathways involved in 4E). Under similar circumstances, integrin-␣v3-blocking CTGF function, we used western blotting to detect the antibody diminished the CTGF-induced ERK1/2 activation in phosphorylation status of Akt and mitogen-activated protein MCF-7/CTGF cells (Fig. 4F). Altogether, these results indicate kinases, which are crucial signaling pathways in cancer that the integrin-␣v3–ERK1/2 pathway is crucial for CTGF- progression (Sebolt-Leopold and Herrera, 2004; Vivanco and induced ERK1/2 activation and subsequent cellular migration. Sawyers, 2002). The phosphorylation status of most of the Activated ERK1/2 regulates membrane protrusions and kinases was not altered, whereas ERK1/2 phosphorylation was focal adhesion turnover through various substrates, such as enhanced in MCF-7/CTGF cells and decreased in cytoplasmic proteins – including MCLK, paxillin and FAK – MDA231/AS cells (Fig. 4A). To explore whether ERK1/2 is or nuclear proteins – including Elk-1 and Ets (Deak et al., involved in CTGF-induced cell migration, we used the MEK1- 1998; Frodin and Gammeltoft, 1999; Fukunaga and Hunter, specific inhibitor PD98059 and a constitutively active MEK1 1997; Hunger-Glaser et al., 2003; Klemke et al., 1997; mutant to modulate the ERK1/2 phosphorylation status. The Waskiewicz et al., 1997). Therefore, we examined the Journal of Cell Science
Fig. 2. CTGF expression promotes the dynamic regulation of actin structures and focal contact sites in breast cancer cells. Distribution of F- actin and paxillin-containing focal adhesions in CTGF-induced morphological alterations. Serum-starved MCF-7/neo, MCF-7/CTGF, MDA231/neo, and MDA231/AS cells were fixed in 3.7% paraformaldehyde and photographed under a light microscope (a,e,i,m). The fixed cells were co-stained with Texas-Red-phalloidin for F-actin (red; b,f,j,n) and monoclonal antibody against paxillin (green; c,g,k,o). In overlays (d,h,c,p), arrows indicate the sites of paxillin, the borders of F-actin colocalization appear in yellow. CTGF promotes breast cancer metastasis 2057 Journal of Cell Science Fig. 3. CTGF-mediated outside-in signaling confers enhanced migratory ability in human breast cancer cells. (A) Effect of rCTGF on the migratory ability of MCF-7 cells. The migratory ability was measured by using a Boyden chamber assay with rCTGF or without (solvent control). Columns show the mean of three independent experiments, and error bars are the corresponding upper 95% confidence intervals. Asterisks denote a statistically significant difference in the migratory ability of treated cells compared with that of control cells (*P<0.05, **P<0.01, two-tailed Student’s t test). (B) Conditioned medium (C.M.) was collected from MDA-MB- 231 cells 48 hours after plating. The migratory ability of MCF-7 cells was measured by Boyden chamber assay with CTGF-neutralizing antibody or without (normal rabbit IgG) of a CTGF-neutralizing antibody. Columns show the mean of three independent experiments, and the error bars are the corresponding upper 95% confidence intervals. Asterisks denote a statistically significant difference in migratory ability of treated cells as compared to control cells (*P<0.05, **P<0.01, two-tailed Student’s t-test, a, differences in migratory ability between treated cells and untreated cells, b, differences in migratory ability between CTGF-neutralized antibody-treated cells and normal rabbit IgG-treated cells). (C) Blockade of CTGF outside-in signaling inhibits the migratory ability of MDA-MB-231. The migratory ability of MDA-MB-231 cells was measured by using a Boyden chamber assay with CTGF-neutralizing antibody or without (normal rabbit IgG). Columns show the mean of three independent experiments, error bars are the corresponding upper 95% confidence intervals. Asterisks denote a statistically significant difference in migratory ability of treated cells as compared to control cells (*P<0.05, ** P<0.01, two-tailed Student’s t-test). (D) Blockade of CTGF outside-in signaling result in morphological alterations and cytoskeleton rearrangements. Serum-starved MDA-MB-231 cells were untreated or treated with CTGF- neutralized antibody or isotype control (normal rabbit IgG). Cells were fixed in 3.7% paraformaldehyde and photographed under a light microscopy (a,e,i). The fixed cells were co-stained with Texas-Red-phalloidin for F-actin (red; b,f,j) and monoclonal antibody against paxillin (green; c,g,k); overlays are shown in d,h,c. (E) CTGF expression resulted in enhanced migratory ability through integrin ␣v3. Cells were pretreated with integrin ␣v3-blocking antibody or without (normal rabbit IgG) or for 6 hours prior the treatment with rCTGF. The migratory ability of MCF-7 cells was measured by the Boyden chamber assay. Columns show the mean of three independent experiments, error bars are the corresponding upper 95% confidence intervals. Asterisks denote statistically significant difference in migratory ability of treated-cells compared with that of control cells (*P<0.05, ** P<0.01, two-tailed Student’s t-test, a, differences in migratory ability between rCTGF-treated cells and untreated cells, b, differences in migratory ability between CTGF-neutralized antibody-treated cells and IgG-treated cells). 2058 Journal of Cell Science 120 (12)
Table 1. Metastasis-associated genes downregulated and E-cadherin levels by RT-PCR and western blotting. The (>fourfold reduction) by AS-CTGF in MDA231 cells expression of E-cadherin mRNA (data not shown) and protein Gene name GenBank Acc. No. was undetectable both in MDA231/neo and MDA231/AS cells (supplementary material Fig. S2); moreover, E-cadherin level CDH1 NM_004360 ANXA8 NM_001630 was not altered under ectopic expression of CTGF in MCF-7 ITGB3 NM_000212 cells (supplementary material Fig. S2), indicating that the AREG NM_001657 deregulation of E-cadherin mRNA in microarray data (Table TFF2 NM_005423 1) is a false-positive and without biological significance. CDH11 NM_033664 S100A4 NM_002961 However, both the mRNA and protein expression levels of MMP15 NM_002428 S100A4 were upregulated in MCF-7/CTGF cells but ITGB4 NM_000213 downregulated in MDA231/AS cells (Fig. 5B). We also found NRG1 NM_013958 that S100A4 was overexpressed in MDA231 and MDA435 IL8RB NM_001557 cells, suggesting a strong association with CTGF expression KIT NM_000222 PDGFRA NM_006206 and cellular aggressiveness (supplementary material Fig. S3). PTGES NM_004878 Subsequently, treatment with the MEK1 inhibitor PD98059 CSF1 NM_000757 effectively abolished the CTGF-induced S100A4 upregulation TGFA NM_003236 (Fig. 5C), whereas transfection with the constitutively active MEK1 mutant significantly increased S100A4 expression in subcellular localization of phosphorylated (P)-ERK1/2 using MDA231/AS cell (Fig. 5D). These results strongly suggest that western blotting and immunofluorescence staining. CTGF CTGF-mediated S100A4 upregulation depend on the ERK1/2 overexpression promoted their activation and nuclear signaling pathway. translocation in MCF-7 cells (Fig. 4G,H), while consistently To ascertain the role of S100A4 in CTGF-mediated cell decreasing the levels of P-ERK1/2 in the nucleus of migration, we generated the pcDNA4-S100A4 and pcDNA4- MDA231/AS cells (Fig. 4G,H). Thus, CTGF can promote AS-S100A4 constructs to establish the doubly transfected ERK1/2 phosphorylation and nuclear translocation, which then stable transfectants of MDA231/AS and MCF-7/CTGF cells, may activate gene expression. respectively. S100A4 expression levels were reduced by AS-
S100A4 as a downstream effecter of CTGF Fig. 4. ERK1/2 is activated by CTGF via integrin ␣v3 and confers The question remained as to which gene was the possible enhanced migratory ability. (A) Effect of CTGF on the activation of downstream effecter that contributed to CTGF-mediated Akt and MAPKs. Cells were serum-starved for 24 hours; activated ERK1/2-dependent cell migratory effect. Since CTGF induced level of Akt and MAPKs were measured by western blotting with P-ERK1/2 nuclear translocation, transcriptional regulation phosphorylation-specific antibodies. (B) The role of P-ERK1/2 in might occur. We used a cDNA microarray to identify the CTGF-mediated cellular migration. Migration assays were genetic expression profile (Tables 1 and 2). S100A4 (Fig. 5A) performed as described above, MCF-7/neo and MCF-7/CTGF cells were treated with dimethylsulfoxide or PD98059 (20 M), whereas
Journal of Cell Science and E-cadherin are of particular interest because they function cells were seeded on the upper chambers (lower figure). Cell lysates as regulators of metastasis of human tumors, and have been were simultaneously analyzed by western blotting with antibodies implicated in cytoskeleton-membrane interaction, migratory against P-ERK1/2 and ERK1 (upper panel). (C) MDA231/neo and behaviors and malignancy in cancer cells (Cho et al., 2003; Cui MDA231/AS cells were transiently transfected active MEK1, 48 et al., 2004; Davies et al., 1996; Ebralidze et al., 1989; Glenney, hours after transfection, cells were trypsinized and assayed using a Jr et al., 1989; Hernan et al., 2003; Jiang, 1996; Kim and Boyden chamber (lower figure). Simultaneously, cell lysates were Helfman, 2003; Masiakowski and Shooter, 1988; analyzed by western blotting with antibodies against P-ERK1/2 and Mazzucchelli, 2002; Missiaglia et al., 2004; Nikitenko et al., ERK1/2 (upper panel). (D) Effect of rCTGF on ERK1/2 activation in 5 2000; Platt-Higgins et al., 2000; Rudland et al., 2000). To MCF-7 cells. Wild-type MCF-7 cells (5ϫ10 ) were seeded in 6-cm confirm the expression levels of S100A4 and E-cadherin in the dishes, serum-starved for 24 hours and then treated with 50 ng/ml stable transfactants and their control cells, we assayed S100A4 rCTGF for 20 minutes to 24 hours. P-ERK1/2 and ERK1 levels were detected by western blotting. (E) Wild-type MCF-7 cells (5ϫ105) were seeded in 6-cm dishes. Cells were serum-starved, pretreated Table 2. Metastasis-associated genes upregulated with 5 ug/ml IgG or integrin-␣v3-blocking antibodies for 24 hours (>fourfold induction) by AS-CTGF in MDA231 cells and then treated with 50 ng/ml rCTGF for 10 minutes. P-ERK1/2 and ERK1 were detected by western blotting. (F) p-ERK1/2 and Gene name GenBank Acc. No. ERK1 expression in MCF-7/neo and MCF-7/CTGF cells treated with MYO1D NM_015194 integrin-␣v3-blocking antibody. Cells were treated with 5 ug/ml FGF13 NM_004114 IgG or integrin-␣v3-blocking antibodies for 24 hours and P- F3 NM_001993 ERK1/2 and ERK1 levels was measured by Western blotting. PCDH7 NM_002589 LDB2 NM_001290 (G) Subcellular localization of CTGF-activated ERK1/2 analyzed by NGFR NM_002507 western blotting. Cells were serum-starved for 24 hours, and the MGMT NM_002412 nuclear and cytosolic fractions were isolated (see Materials and IL8 NM_000584 Methods); P-ERK1/2 and ERK1 were detected by western blotting. CCNG2 NM_004354 (H) Subcellular localization of CTGF-induced P-ERK1/2 was IL1RN NM_173841 analyzed by immunofluorescence staining. Cells were fixed in 3.7% TPD52L1 NM_001003395 paraformaldehyde and co-stained with anti-P-ERK1/2 antibody and RASSF2 NM_014737 DAPI. Arrows indicate increased expression of CTGF-induced P- IL11RA NM_147162 ERK1/2 in nuclei of breast cancer cells. CTGF promotes breast cancer metastasis 2059 Journal of Cell Science
Fig. 4. See previous page for legend. 2060 Journal of Cell Science 120 (12) Journal of Cell Science
Fig. 5. See next page for legend. CTGF promotes breast cancer metastasis 2061
Table 3. Regulation of lung metastasis in nude mice by the CTGF-S100A4 pathway Lung weight (mg) Number of mice with lung Mean lung Cell lines Mean ± s.d. metastasis vs total number of mice nodules (range) MDA231/neo/pcDNA4 468±69 6/10 39 (0-58) MDA231/neo/S100A4 472±60 7/10 33 (0-61) MDA231/AS/pcDNA4 424±41 4/10 9 (0-26)a,** MDA231/AS/S100A4 461±56 6/10 28 (0-55)b,**
aMDA231/AS/pcDNA4 versus MDA231/neo/pcDNA4 by the Student’s t-test; bMDA231/AS/S100A4 versus MDA231/neo/pcDNA4 by the Student’s t-test, **P<0.01.
S100A4 in MCF-7/CTGF cells, which also decreased the were higher than those in MDA231/AS/pcDNA4-bearing mice migratory ability (Fig. 5E). Restoring levels of S100A4 in (Table 3). In the MDA231/AS/S100A4 group, the mean lung MDA231/AS cells significantly enhanced the migratory ability nodule number was 24 (Table 3), indicating that transfection (Fig. 5F). Immunofluorescence staining further showed that with S100A4 dramatically increased the mean number of transfection with S100A4 significantly promoted re- metastatic nodules and the occurrence of lung metastasis in polymerization of actin stress fibers and aggregation of focal MDA231/AS tumor-bearing mice. Together, these data adhesion complexes in MDA231/AS cells (Fig. 5G). These demonstrate that CTGF-mediated tumor metastasis requires results indicate that S100A4 is a crucial downstream effecter the expression of S100A4 involved in CTGF-mediated cytoskeleton rearrangement and cellular migration. CTGF expression levels are correlated with S100A4 To investigate whether the CTGF-induced S100A4 expression levels in primary human breast cancer expression plays a causal role in tumor metastasis, we injected To establish whether expression levels of CTGF and S100A4 MDA231/neo/pcDNA4, MDA231/neo/S100A4, MDA231/AS/ are correlated in clinical breast cancer, 24 primary human pcDNA4, and MDA231/AS/S100A4 cells intravenously into breast cancer specimens and their non-tumor counterparts were the tail vein of nude mice and measured the metastatic collected, and analyzed semi-quantitatively by RT-PCR and colonization. Whereas tumors expressing the AS-CTGF quantitatively by densitometry (Fig. 6). Non-tumor cells formed very few macroscopically visible metastases in the displayed extremely low or undetectable levels of CTGF and lungs, tumors that co-expressed AS-CTGF and S100A4 S100A4 expression. Conversely, all tumor tissues expressed formed a large number of metastases (Fig. 5H). In the both CTGF and S100A4 (Fig. 6A). Based on our observations MDA231/neo/pcDNA4 group, the mean lung nodule number in breast cancer cells, the expression levels of CTGF and was 39; in the MDA231/AS/pcDNA4 group, the mean lung S100A4 were found to closely correlate in-patient samples nodule number was 9. The numbers of visible metastatic (Fig. 6B, r2=0.65, P=0.0012). These data strongly support the nodules in mice injected with MDA231/neo/pcDNA4 cells hypothesis that CTGF signaling upregulates the expression of
Journal of Cell Science S100A4 in primary human breast cancer. Fig. 5. S100A4 acts as a crucial downstream effecter of CTGF. (A) cDNA microarray analysis (Cy3-Cy5 merged image) showing Discussion the expression of S100A4, arrow indicates human S100A4. In this study, we demonstrate that the molecular mechanism by (B) Expression of S100A4 mRNA and protein by RT-PCR and which CTGF-confers cellular metastatic ability is mediated by western blotting, respectively. (C) Effects of ERK1/2 activation on S100A4 upregulation by integrin ␣v3 and/or ERK1/2. This CTGF-regulated S100A4 expression. MCF-7/neo and MCF-7/CTGF is based on the following evidence. First, overexpression of cells were treated with DMSO or PD98059 (20 M) for 24 hours, CTGF appreciably increases the migratory ability of MCF-7 and the levels of S100A4 were evaluated by western blotting. cells. Conversely, knockdown of CTGF abolishes the (D) MDA231/neo and MDA231/AS cells were transiently migratory ability of MDA231 cells. Second, CTGF expression transfected by constitutively activated MEK1; 48 hours after leads to morphological alterations and formation of F-actin and transfection, expression levels of S100A4 were evaluated by western blotting. (E) Effects of S100A4 on CTGF-mediated cellular motility. focal adhesions. Third, ERK1/2 activation is essential for the MCF-7/neo and MCF-7/CTGF cells were stably transfected with CTGF-mediated migratory effects. Fourth, blockade of the pcDNA4 or pcDNA4-AS-S100A4, respectively (see Materials and CTGF–integrin-␣v3 axis attenuates CTGF-induced ERK1/2 Methods). Expression levels of S100A4 were detected by western activation and subsequent cellular migration. Fifth, the blotting (upper panel). Cellular motility was measured by migration prometastatic S100A4 gene is regulated by the signaling assay (lower panel). (F) MDA231/neo and MDA231/AS cells were cascades of CTGF–integrin-␣v3–ERK1/2 and contributes to stably transfected with pcDNA4 or pcDNA4-S100A4, respectively. the metastatic ability. Finally, CTGF expression levels are Expression levels of S100A4 were detected by western blotting correlated with S100A4 expression levels in primary human (upper panel). Cellular motility was measured by migration assay breast tumors. (lower panel). (G) S100A4 is involved in CTGF-mediated When CTGF-overexpressing cells were treated with the cytoskeletal changes. Cells were fixed in 3.7% paraformaldehyde and photographed under a light microscope (a,d,g,i). The fixed cells were MEK1 inhibitor PD98059 their migratory ability was inhibited. co-stained with Texas-Red-phalloidin for F-actin (red; b,e,h,k) and Moreover, constitutively active MEK1 also prevented the monoclonal antibody against paxillin (green; c,f,i,l). (H) S100A4 migratory ability in MDA231/AS cells. Thus, our data suggest expression is required for CTGF-mediated metastatic colonization. that the ERK1/2 signaling pathway is crucial for CTGF- Lungs of female BALE/cAnN-Foxn1nu/CrlNarl nude mice were induced cell migration. In support of our observations, others excised and photographed after the experimental metastasis assay. have demonstrated that constitutive activation of ERK1/2 2062 Journal of Cell Science 120 (12)
predominantly located in the nucleus, indicating that CTGF- mediated signaling is possibly regulated by ERK1/2-dependent transcriptional regulation, rather than by paxillin-FAK interaction in breast cancer cells. Since CTGF expression promotes ERK1/2 activation and subsequent cellular migration, we investigated the origin of the CTGF-mediated signaling pathway. Although CTGF is a secreted protein, it carries out its biological activities via intracellular transport (Wahab et al., 2001). Thus, clarification of the origin of CTGF-mediated signaling is important for further in vitro studies that may contribute to therapeutic approaches. According to our data, rCTGF rapidly and transiently induces ERK1/2 phosphorylation. Moreover, CTGF-neutralizing antibody and integrin-␣v3-blocking antibody are dose dependently inhibitory. These results indicate that the outside-in signaling is important for CTGF- mediated biological activities in breast cancer cells. CTGF exerts a range of diverse functions, many of which involve the binding to cell-surface integrins, such as integrins ␣v3, ␣IIb3, ␣M2 and ␣51 (Babic et al., 1999; Jedsadayanmata Fig. 6. CTGF expression levels are correlated with S100A4 et al., 1999; Schober et al., 2002; Weston et al., 2003). Of those, expression levels in samples of primary human breast cancer. integrin ␣v3 is the only integrin highly correlated with breast (A) RT-PCR analysis of CTGF and S100A4 expression in nine cancer metastasis and its progression (Taddei et al., 2003). representative samples of 24 analyzed primary breast cancer samples. CTGF induces adhesion and activation of rat hepatic stellate N, non-tumor counterparts; T, tumor tissue. (B) Graph depicts the cells by C-terminally binding to integrin ␣v3 (Gao and significant correlation between CTGF and S100A4 transcript levels Brigstock, 2004). Moreover, this integrin, which is important determined in all 24 breast cancer samples by densitometric analysis 2 in osteoclast attachment to bone, is highly expressed in breast and normalized to GAPDH control (r =0.65, P=0.0012). cancer cells and bone (Liapis et al., 1996). Although antibody against integrin ␣v3 interfered with CTGF-induced ERK1/2 activity and migration, it is possible that integrin-␣v3- frequently occurs in a variety of cancers, including lung cancer, mediated adhesion is required as a parallel pathway for cervical cancer and breast tumors (Adjei, 2005; Branca et al., ERK1/2 activity and migration. Additionally, ectopic 2004; Milde-Langosch et al., 2005). Constitutive ERK1/2 expression of CTGF induced integrin 3 expression activation has also been observed in different breast cancer (supplementary material Fig. S1A) and integrin ␣v3
Journal of Cell Science cells (Santen et al., 2002), and activation of ERK1/2 leads to formation in MCF-7 cells (supplementary material Fig. cell migration (Krueger et al., 2001). Mutationally activated S1B,C). Also, knockdown of CTGF reduced expression of receptor tyrosine kinases – such as Ras – or binding of growth integrin 3 (Table 1 and supplementary material Fig. S1A) and factors – such as transforming growth factor alpha (TGF␣), formation of integrin ␣v3 in MDA231 cells (supplementary epidermal growth factor (EGF), vascular endothelial growth material Fig. S1B,C). This regulation suggests an amplification factor-A (VEGF-A), platelet-derived growth factor beta loop within the CTGF–integrin-␣v3 signaling pathway (PDGF) and heregulin – to their cognate receptors may induced by CTGF expression. All of the above suggests that induce ERK1/2 activation (Seton-Rogers et al., 2004; Gollob the integrin ␣v3 is the most crucially involved in breast et al., 2006). We presently found that CTGF induces cell cancer metastasis. Our study also shows that the CTGF- migration in an ERK1/2-dependent manner. Thus, ERK1/2 mediated signaling pathway and the cell migratory effects are signaling, which is vital for breast cancer cell migration, is mainly activated through integrin ␣v3. regulated by CTGF and contributes to the increased migratory S100A4, the crucial molecule downstream the CTGF- ability. Activated ERK1/2 regulates membrane protrusions and mediated signaling pathway, is closely associated with focal adhesion turnover via various substrates. The cytoplasmic metastasis in other breast cancer cells, as well as in rodent and substrates of P-ERK1/2 include several protein kinases, such in human cancer specimens (Ambartsumian et al., 1996; as RSK1, MSK1, MNK1/2, myosin light chain kinase (MCLK) Ambartsumian et al., 2001; Cho et al., 2003; Cui et al., 2004; and FAK (Deak et al., 1998; Frodin and Gammeltoft, 1999; Hernan et al., 2003; Mazzucchelli, 2002; Missiaglia et al., Fukunaga and Hunter, 1997; Hunger-Glaser et al., 2003; 2004; Platt-Higgins et al., 2000). Elevated levels of S100A4 Klemke et al., 1997; Waskiewicz et al., 1997), the protease have been found in metastatic cancers in mice and in humans calpain (Glading et al., 2004) and paxillin (Liu et al., 2002). (Ebralidze et al., 1989; Nikitenko et al., 2000). Its expression However, ERK1/2 translocation into the nucleus after is also strongly correlated with the demise of breast cancer activation contributes to transcriptional regulation. The key patients (Rudland et al., 2000). Increased levels of rat or human mechanism is believed to involve the further phosphorylation S100A4 result in metastatic capability of initially benign and activation of key transcription factors such as Ets, CREB, mammary tumor cells in rats (Ambartsumian et al., 1996; Jun, and Myc (Santen et al., 2002). These events are likely to Davies et al., 1993; Davies et al., 1996; Lloyd et al., 1998). be mediated through stimulation of transcription in the nucleus. Although the precise function of S100A4 is not entirely clear, In our study, CTGF-mediated ERK1/2 phosphorylation is its interaction with cytoskeletal moieties and its early role in CTGF promotes breast cancer metastasis 2063
EMT indicates that S100A4 expression is involved in the replated in MEM containing 10% FBS 800 g/ml G418. G418-resistant clones (MCF-7/neo, MCF-7/CTGF, MDA231/neo, and MDA231/AS) were selected and migratory process (Okada et al., 1997). This may result from pooled for further studies. Doubly transfected cells harboring another pcDNA4- its role in the reorganization of cytoskeletal components, such based plasmid were trypsinized after 48 hours of transfection, and replated in MEM as nonmuscle myosin II (Ford et al., 1995; Kriajevska et al., supplemented with 10% FBS, 50 g/ml G418 and 500 ug/ml zeocin. Zeocin- 1994), actin (Watanabe et al., 1993) and tropomycin (Takenaga resistant clones were selected, expanded and pooled for further studies. et al., 1994), which indicates that S100A4 is involved in Western blotting motility by interacting with various components of the Proteins in the total cell lysate (40 g of protein) were separated on 10% SDS- cytoskeleton (Okada et al., 1997). In addition, S100A4 can act PAGE and electrotransferred to a polyvinylidene difluoride membrane (Immobilon- as an angiogenic factor (Ambartsumian et al., 2001), and can P membrane; Millipore, Bedford, MA). After the blot was blocked in a solution of 5% skimmed milk, 0.1% Tween 20 and PBS, membrane-bound proteins were negatively regulate bone mineralization and osteoblast probed with primary antibodies against CTGF, S100A4, P-ERK1/2, ERK1/2, SP1 differentiation (Duarte et al., 2003). In endothelial cells, (Santa Cruz Biotechnology, Santa Cruz, CA). The membrane was washed and then S100A4 activates transcription of matrix metallopeptidases incubated with horseradish peroxidase-conjugated secondary antibodies for 30 minutes. Antibody-bound protein bands were detected with enhanced MMP11, MMP13 and MMP14 (Schmidt-Hansen et al., 2004a; chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ) and Schmidt-Hansen et al., 2004b). Accordingly, we hypothesize photographed with Kodak X-Omat Blue autoradiography film (Perkin Elmer Life that CTGF-regulated S100A4 expression not only confers Sciences, Boston, MA). increased metastasis, but also regulates angiogenesis, bone homeostasis and the transcription of MMPs. Together, our RNA isolation and RT-PCR Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA) according to the study and studies by others provide the evidence that S100A4 manufacturer’s instructions, and 1 g was reverse transcribed into single-stranded is a crucial factor in modulating motility in human breast cDNA with M-MLV reverse transcriptase and random hexamers (Promega, cancer cells, and that its expression can be regulated by CTGF. Madison, WI). Amplification of cDNAs was performed by PCR with specific primer pairs. Primers were, for CTGF 5Ј-ACTGTCCCGGAGACAATGAC-3Ј (forward) In conclusion, we provide a detailed mechanism describing and 5Ј-TGCTCCTAAAGCCACACCTT-3Ј (reverse), for S100A4 5Ј-TCTCTCC - the potential role of CTGF in migratory behavior via an TCAGCGCTTCTTC-3Ј (forward) and 5Ј-CTTCCTGGGCTGCTTA TCTG-3Ј integrin-␣v3–P-ERK1/2-dependent upregulation of S100A4. (reverse), for GAPDH 5Ј-ACCCAGAAGACTGTGGATGG-3Ј (forward) and 5Ј- GTCCACCACCCTGTTGCTGT-3Ј (reverse). PCR conditions were: denaturing This is the first identification of a new molecular mechanism once at 95°C (10 minutes), 95°C (1 minute), 52°C (1 minute) and 72°C (1 minute) regulating the CTGF-driven prometastatic pathway, possibly for 30 cycles, once at 72°C (10 minutes). PCR products were analysed using agarose providing a new target for therapeutic intervention in gel electrophoresis. metastatic breast cancer. Immunofluorescence staining For immunofluorescence staining, all fixation and staining procedures were Materials and Methods conducted at room temperature. Cultures were grown in a Lab-Tek four-chamber Cell culture, antibodies and reagents slide apparatus (Nalge NUNC, Rochester, NY), fixed with cold 4% The human breast cancer cell lines MCF-7 and MDA231 were obtained from the paraformaldehyde for 15 minutes, washed three times with phosphate-buffered American Type Culture Collection. BT474, BT483, MDA-MB-453, MDA231 and saline (PBS) and permeabilized with 0.1% Triton X-100 in PBS for 5 minutes. After MDA-MB-435 cells were provided generously by the National Health Research washing three times with 0.05% Tween 20 in PBS (PBST), cultures were blocked Institute (Taipei, Taiwan). These cell lines were maintained in minimum essential in 5% nonfat milk (Carnation) in PBST for 30 minutes. Cells were incubated with medium (MEM) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, primary antibodies against paxillin or P-ERK1/2) for 1 hour and incubated at room Gaithersburg, MD), 2 mM L-glutamine (Life Technologies, Carlsbad, CA), temperature with Texas-Red-conjugated phalloidin (Molecular Probes) as specified Journal of Cell Science 100 g/ml streptomycin and 100 U/ml penicillin in a humidified 5% CO2 in each experiment. After washing, the cells were incubated with secondary atmosphere. Monoclonal mouse anti-human CTGF antibody was purchased from antibody (1:100) and DAPI (1:5000) for 60 minutes. The control samples included R&D Systems (Minneapolis, MN). Antibodies against integrin ␣v3, ␣-tubulin, P- those treated with either secondary antibody alone or with pre-immune mouse ERK1/2 and ERK1/2 were purchased from Santa Cruz Biotechnology (Santa Cruz, serum. The slides were mounted with VectorShield (Vector Laboratories, CA). Polyclonal rabbit anti-human S100A4 antibody was purchased from Abnova Burlingame, CA) and examined under a Nikon Eclipse E600 upright microscope Biotechnology (Taipei, Taiwan). Anti-paxillin antibody was from BD Biosciences equipped with fluorescent devices. (Franklin Lakes, NJ). Goat anti-rabbit or anti-mouse IgG conjugated with either fluorescein isothiocynate (FITC) or tetramethylrhodamine isothiocyanate (TRITC) Northern blot analysis were purchased from Jackson Immunoresearch (West Grove, PA). Diamidino Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA) according to the phenylindole dimethylsulfoxide (DAPI) was purchased from Molecular Probes manufacturer’s instructions and 10 g were separated by electrophoresis on 1.2% (Eugene, OR). The constitutively active MEK1 mutant construct (substitution of the agarose. RNA was transferred to Hybond-XL membranes (Amersham Pharmacia regulatory phosphorylation sites S218D and S222D) as described previously Biotech, Piscataway, NJ), then hybridized with cDNA probe and randomly labeled (Brunet et al., 1994) was gift from Ruey-Hwa Chen. with ␣[32P]dATP (3000 Ci/mmol/l; DuPont/NEN, Boston, MA). Probes were labeled using the Prime-It II Random Primer kit (Stratagene, La Jolla, CA). Boyden chamber migration assays Membranes were stripped and reprobed with cDNA for GAPDH mRNA and Migration of human breast cancer cells through polycarbonate filters was examined exposed on Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY) was exposed in 24-well modified Boyden chambers (pore size 8 m). The lower wells of the for various periods of time to the blots. chamber were loaded with 1 ml of MEM. Breast cancer cells (50,000 cells per 100 l) were placed in the upper wells. After 6 hours of incubation, cells on the lower Flow cytometry surface of the filter were fixed and stained with Crystal Violet and counted using a Cells were washed once with PBS and harvested in 0.05% trypsin/0.025% EDTA. microscope (type 090-135.001; Leica Microsystems, Wetzlar, Germany). Detached cells were washed with PBS containing 1% FCS and 1% penicillin- Incubations were performed in triplicate and each experiment was repeated at least streptomycin (wash buffer), and resuspended in wash buffer (106 cells per 100 l). three times. Cells were then incubated with anti-integrin ␣v3 monoclonal antibodies (Santa Cruz Biotechnology) or respective isotype controls and FITC-conjugated secondary Establishment of stably transfected cells antibodies (Sigma-Aldrich) for 30 minutes on ice. The labeled cells were washed A 1.2-kb pair sense- or antisense-orientation cDNA fragment of human CTGF was in wash buffer, fixed in PBS containing 1% paraformaldehyde and then analyzed each cloned into the vector pcDNA3 to produce sense- and antisense-oriented CTGF on a FACSVantage (BD Biosciences). expression vectors. Furthermore, 0.4 kb sense- or antisense cDNA fragments of human S100A4 were each cloned into the vector pcDNA4 to produce sense-oriented Microarray analysis (pcDNA4-S100A4) and antisense-oriented (pcDNA4-AS-S100A4) S100A4 The Agilent human 1 cDNA microarray (Agilent Technology) containing 18,564 expression vectors. Cells were transfected with plasmids using the lipofectin spots of 13,574 different genes was used in this study. Fifteen micrograms of purified transfection reagent (Invitrogen, Carlsbad, CA). Transfected cells were grown in an total RNA were converted to cDNA using a 3DNATM Array 50 Expression Array atmosphere of 5% CO2 at 37°C in MEM supplemented with 10% FBS. After 48 Detection Kit (Genisphere). RNA was labeled with Cy3, and RNA from Universal hours of transfection with pcDNA3-based plasmids, cells were trypsinized and Human Reference RNA was labeled with Cy5. Correspondingly synthesized cDNA 2064 Journal of Cell Science 120 (12)
products were combined and concentrated by ethanol precipitation and suspended in metastasis-associated Mts1 (S100A4) protein could act as an angiogenic factor. hybridization buffer. Labeled cDNA was hybridized to Agilent human 1 cDNA Oncogene 20, 4685-4695. microarray (Agilent Technologies) at 65°C for 17 hours. After hybridization, slides Babic, A. M., Chen, C. C. and Lau, L. F. (1999). Fisp12/mouse connective tissue growth were washed in 5ϫSSC with 0.01% SDS at room temperature for 5 minutes and factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, 0.06ϫSSC at room temperature for 2 minutes. Washed microarrays were then promotes endothelial cell survival, and induces angiogenesis in vivo. Mol. Cell. Biol. hybridized with Cy3 and Cy5 dendrimers in formamide-based buffer at 53°C for 3 19, 2958-2966. Barrett-Lee, P. J. (2005). Growth factor signalling in clinical breast cancer and its impact hours. After hybridization with dendrimers, slides were washed in 2ϫSSC with ϫ on response to conventional therapies: a review of chemotherapy. Endocr. Relat. 0.01% SDS at 42°C for 15 minutes, 2 SSC at room temperature for 10 minutes, Cancer. 12 Suppl. 1, S125-S133. ϫ and 0.2 SSC at room temperature for 10 minutes. Washed microarrays were scanned Berry, M. G., Gui, G. P., Wells, C. A. and Carpenter, R. (2004). Integrin expression with a Virtek fluorescence reader (Virtek, CA) at 532 nm and 635 nm for Cy3 and and survival in human breast cancer. Eur. J. Surg. Oncol. 30, 484-489. Cy5, respectively. Scanned images were analyzed by Array-Pro image acquisition Bork, P. (1993). The modular architecture of a new family of growth regulators related software (Media Cybernetics); an image analysis algorithm was used to quantify to connective tissue growth factor. FEBS Lett. 327, 125-130. signal and background intensity for each target spot (gene). The mean intensity of Branca, M., Ciotti, M., Santini, D., Bonito, L. D., Benedetto, A., Giorgi, C., Paba, P., the spot area was computed as the microarray raw data. The data were further Favalli, C., Costa, S., Agarossi, A. et al. (2004). Activation of the ERK/MAP kinase analyzed with software package S-Plus 6.1 (Venables, 2002). pathway in cervical intraepithelial neoplasia is related to grade of the lesion but not to high-risk human papillomavirus, virus clearance, or prognosis in cervical cancer. Am. J. Clin. Pathol. 122, 902-911. Experimental metastasis Brunet, A., Pages, G. and Pouyssegur, J. (1994). Constitutively active mutants of MAP Cells were washed and resuspended in PBS. Subsequently, a single-cell suspension 6 kinase kinase (MEK1) induce growth factor-relaxation and oncogenicity when containing 10 cells in 0.1 ml of PBS was injected into the lateral tail vein of 7- expressed in fibroblasts. Oncogene 9, 3379-3387. week-old female nude mice (supplied by the animal center in the College of Brunton, V. G., MacPherson, I. R. and Frame, M. C. (2004). Cell adhesion receptors, Medicine, National Taiwan University, Taipei, Taiwan). Mice were killed after 18 tyrosine kinases and actin modulators: a complex three-way circuitry. Biochim. weeks. (Our preliminary study in this animal model indicated that MDA231 cells Biophys. Acta 1692, 121-144. developed numerous lung metastasis nodules by 18 weeks.) All organs were Cho, Y. G., Nam, S. W., Kim, T. Y., Kim, Y. S., Kim, C. J., Park, J. Y., Lee, J. H., examined for metastasis formation. Lungs were removed, weighed and fixed in 10% Kim, H. S., Lee, J. W., Park, C. H. et al. (2003). Overexpression of S100A4 is closely formalin. Lung tumor colonies were counted under a dissecting microscope. All related to the aggressiveness of gastric cancer. APMIS 111, 539-545. animal work was performed usinmg protocols approved by the Institutional Animal Cui, J. F., Liu, Y. K., Pan, B. S., Song, H. Y., Zhang, Y., Sun, R. X., Chen, J., Feng, Care and Use Committee of the College of Medicine, National Taiwan University. J. T., Tang, Z. Y., Yu, Y. L. et al. (2004). Differential proteomic analysis of human hepatocellular carcinoma cell line metastasis-associated proteins. J. Cancer Res. Clin. Oncol. 130, 615-622. Wound healing assay Damjanovich, L., Fulop, B., Adany, R. and Nemes, Z. (1997). Integrin expression on Cell monolayers were wounded 24 hours after plating by scratching with a pipette normal and neoplastic human breast epithelium. Acta Chir. Hung. 36, 69-71. tip. Debris was removed by washing. Images were taken at 20ϫ magnification Darash-Yahana, M., Pikarsky, E., Abramovitch, R., Zeira, E., Pal, B., Karplus, R., (Nikon Ph1 DL; NA 0.4) with a Nikon TMS microscope equipped with a Nikon F- Beider, K., Avniel, S., Kasem, S., Galun, E. et al. (2004). Role of high expression 601 camera. Distances between wound edges were measured at five sites/image levels of CXCR4 in tumor growth, vascularization, and metastasis. FASEB J. 18, 1240- (n=3). Alternatively, wounded monolayers were subjected to time-lapse studies for 1242. 3 hours at 10ϫ magnification using a Zeiss Axiovert 200 microscope equipped with Davies, B. R., Davies, M. P., Gibbs, F. E., Barraclough, R. and Rudland, P. S. (1993). the AxioCam digital system. Induction of the metastatic phenotype by transfection of a benign rat mammary epithelial cell line with the gene for p9Ka, a rat calcium-binding protein, but not with the oncogene EJ-ras-1. Oncogene 8, 999-1008. Purification of the rCTGF protein Davies, M. P., Rudland, P. S., Robertson, L., Parry, E. W., Jolicoeur, P. and To purify the recombinant CTGF protein (rCTGF) protein, 293T cells were used to Barraclough, R. (1996). Expression of the calcium-binding protein S100A4 (p9Ka) express rCTGF. Protein A Sepharose 4 Fast Flow was used with an Atka-fast protein in MMTV-neu transgenic mice induces metastasis of mammary tumours. Oncogene liquid chromoatography system (Amersham Pharmacia, Freiburg, Germany). 13, 1631-1637. Columns were equilibrated with PBS (pH 7.0), the supernatant (15-45 ml) was Deak, M., Clifton, A. D., Lucocq, J. M. and Alessi, D. R. (1998). Mitogenand stress- applied at a flow rate of 2 ml/minute. Columns were washed with ten column activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38,
Journal of Cell Science volumes of PBS and the protein was eluted with elution buffer (0.1 M glycine pH and may mediate activation of CREB. EMBO J. 17, 4426-4441. 2.5). Directly thereafter, eluted fractions were neutralized using 3 M Tris-HCl pH Duarte, W. R., Shibata, T., Takenaga, K., Takahashi, E., Kubota, K., Ohya, K., 8. The rCTGF protein was desalted using a PD-10 column (Amersham Pharmacia) Ishikawa, I., Yamauchi, M. and Kasugai, S. (2003). S100A4: a novel negative and the purity of the protein checked by silver staining and western blotting. regulator of mineralization and osteoblast differentiation. J. Bone Miner. Res. 18, 493- 501. Ebralidze, A., Tulchinsky, E., Grigorian, M., Afanasyeva, A., Senin, V., Revazova, E. Preparation of CTGF-neutralizing antibody and Lukanidin, E. (1989). Isolation and characterization of a gene specifically Anti-CTGF antibody was raised in rabbits by immunization with a synthetic CTGF expressed in different metastatic cells and whose deduced gene product has a high peptide composed of 20 amino acids (aa 243 to aa 263; EADLEENIKKGKK - degree of homology to a Ca2+-binding protein family. Genes Dev. 3, 1086-1093. CIRTPKIS). This sequence is shared with CTGF (also known as Fisp12 in mouse) Felding-Habermann, B., O’Toole, T. E., Smith, J. W., Fransvea, E., Ruggeri, Z. M., (the mouse homologue of), but not CYR61, NOV, WISP1 (also known as Elm1 in Ginsberg, M. H., Hughes, P. E, Pampori, N., Shattil, S. J., Saven, A. et al. (2001). mouse), WISP2 (also known as rCop1 in mouse) or WISP3. Anti-CTGF antibody Integrin activation controls metastasis in human breast cancer. Proc. Natl. Acad. Sci. was purified from the serum as previously described (Shimo et al., 1999). USA 98, 1853-1858. Ford, H. L., Salim, M. M., Chakravarty, R., Aluiddin, V. and Zain, S. B. (1995). Expression of Mts1, a metastasis-associated gene, increases motility but not invasion We thank the anonymous reviewers for helpful comments. This of a nonmetastatic mouse mammary adenocarcinoma cell line. Oncogene 11, 2067- work was supported by grants from the Department of Industrial 2075. Technology, Ministry of Economic Affairs, Taipei, Taiwan (95-EC- Frodin, M. and Gammeltoft, S. (1999). Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction. Mol. Cell. Endocrinol. 151, 65-77. 17-A-19S1-016), the National Science Council, Taiwan (NSC95- Fukunaga, R. and Hunter, T. (1997). MNK1, a new MAP kinase-activated protein 2314-B-002-318-MY3, NSC94-2320-B-002-012, and NSC94-2323- kinase, isolated by a novel expression screening method for identifying protein kinase B-002-010), and the National Taiwan University Hospital (NTUH- substrates. EMBO J. 16, 1921-1933. 94M04). We thank Tung-Tien Sun (Department of Urology, New York Gao, R. and Brigstock, D. R. (2004). Connective tissue growth factor (CCN2) induces adhesion of rat activated hepatic stellate cells by binding of its C-terminal domain to University School of Medicine, NY) for revising this paper. Ruey- integrin alpha(v)beta(3) and heparan sulfate proteoglycan. J. Biol. Chem. 279, 8848- Hwa Chen is thanked for providing the active MEK1 plasmid. 8855. Glading, A., Bodnar, R. J., Reynolds, I. J., Shiraha, H., Satish, L., Potter, D. A., Blair, H. C. and Wells, A. (2004). Epidermal growth factor activates m-calpain (calpain II), References at least in part, by extracellular signal-regulated kinase-mediated phosphorylation. Mol. Adjei, A. A. (2005). The role of mitogen-activated ERK-kinase inhibitors in lung cancer Cell. Biol. 24, 2499-2512. therapy. Clin. Lung Cancer 7, 221-223. Glenney, J. R., Jr, Kindy, M. S. and Zokas, L. (1989). Isolation of a new member of Ambartsumian, N. S., Grigorian, M. S., Larsen, I. F., Karlstrom, O., Sidenius, N., the S100 protein family: amino acid sequence, tissue, and subcellular distribution. J. Rygaard, J., Georgiev, G. and Lukanidin, E. (1996). Metastasis of mammary Cell Biol. 108, 569-578. carcinomas in GRS/A hybrid mice transgenic for the mts1 gene. Oncogene 13, 1621- Gollob, J. A., Wilhelm, S., Carter, C. and Kelley, S. L. (2006). Role of Raf kinase in 1630. cancer: therapeutic potential of targeting the Raf/MEK/ERK signal transduction Ambartsumian, N., Klingelhofer, J., Grigorian, M., Christensen, C., Kriajevska, M., pathway. Semin. Oncol. 33, 392-406. Tulchinsky, E., Georgiev, G., Berezin, V., Bock, E., Rygaard, J. et al. (2001). The Gui, G. P., Wells, C. A., Yeomans, P., Jordan, S. E., Vinson, G. P. and Carpenter, R. CTGF promotes breast cancer metastasis 2065
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