QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online June 5, 2007
doi: 10.1242/10.1242/jcs.03460

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chen, P.-S. Articles by Kuo, M.-L. Search for Related Content PubMed PubMed Citation Articles by Chen, P.-S. Articles by Kuo, M.-L. Social Bookmarking                         What's this?

CTGF enhances the motility of breast cancer cells via an integrin-vβ3–ERK1/2-dependent S100A4-upregulated pathway

Pai-Sheng Chen^1,2,*, Ming-Yang Wang^1,2,3,*, Shin-Ni Wu¹, Jen-Liang Su^4,5,6, Chih-Chen Hong⁷, Shuang-En Chuang⁷, Min-Wei Chen¹, Kuo-Tai Hua¹, Yu-Ling Wu¹, Shih-Ting Cha¹, Munisamy Suresh Babu¹, Chiung-Nien Chen^2,3, Po-Huang Lee^2,3, King-Jen Chang^2,3,, and Min-Liang Kuo^1,2,,

¹ Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan
² Angiogenesis Research Center, National Taiwan University, Taipei, Taiwan
³ Department of Surgery, National Taiwan University Hospital, Taipei 100, Taiwan
⁴ Graduate Institute of Cancer Biology, College of Medicine, China Medical University, Taichung 404, Taiwan
⁵ Center for Molecular Medical, China Medical University Hospital, Taichung 404, Taiwan
⁶ Department of Biotechnology and Bioinformatics, Asia University, Taichung 41354, Taiwan
⁷ Division of Cancer Research, National Health Research Institutes, Taipei 10016, Taiwan

View larger version (40K): [in this window] [in a new window]   Fig. 1. CTGF expression enhances migratory ability in human breast cancer cells. (A) Expression of CTGF was detected in BT474, BT483, 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 20x 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).

View larger version (82K): [in this window] [in a new window]   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.

View larger version (55K): [in this window] [in a new window]   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 vβ3. Cells were pretreated with integrin vβ3-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).

View larger version (47K): [in this window] [in a new window]   Fig. 4. ERK1/2 is activated by CTGF via integrin vβ3 and confers enhanced migratory ability. (A) Effect of CTGF on the activation of Akt and MAPKs. Cells were serum-starved for 24 hours; activated level of Akt and MAPKs were measured by western blotting with phosphorylation-specific antibodies. (B) The role of P-ERK1/2 in CTGF-mediated cellular migration. Migration assays were performed as described above, MCF-7/neo and MCF-7/CTGF cells were treated with dimethylsulfoxide or PD98059 (20 µM), whereas cells were seeded on the upper chambers (lower figure). Cell lysates were simultaneously analyzed by western blotting with antibodies against P-ERK1/2 and ERK1 (upper panel). (C) MDA231/neo and MDA231/AS cells were transiently transfected active MEK1, 48 hours after transfection, cells were trypsinized and assayed using a Boyden chamber (lower figure). Simultaneously, cell lysates were analyzed by western blotting with antibodies against P-ERK1/2 and ERK1/2 (upper panel). (D) Effect of rCTGF on ERK1/2 activation in MCF-7 cells. Wild-type MCF-7 cells (5x105) were seeded in 6-cm dishes, serum-starved for 24 hours and then treated with 50 ng/ml rCTGF for 20 minutes to 24 hours. P-ERK1/2 and ERK1 levels were detected by western blotting. (E) Wild-type MCF-7 cells (5x105) were seeded in 6-cm dishes. Cells were serum-starved, pretreated with 5 ug/ml IgG or integrin-vβ3-blocking antibodies for 24 hours 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 ERK1 expression in MCF-7/neo and MCF-7/CTGF cells treated with integrin-vβ3-blocking antibody. Cells were treated with 5 ug/ml IgG or integrin-vβ3-blocking antibodies for 24 hours and P-ERK1/2 and ERK1 levels was measured by Western blotting. (G) Subcellular localization of CTGF-activated ERK1/2 analyzed by western blotting. Cells were serum-starved for 24 hours, and the nuclear and cytosolic fractions were isolated (see Materials and Methods); P-ERK1/2 and ERK1 were detected by western blotting. (H) Subcellular localization of CTGF-induced P-ERK1/2 was analyzed by immunofluorescence staining. Cells were fixed in 3.7% paraformaldehyde and co-stained with anti-P-ERK1/2 antibody and DAPI. Arrows indicate increased expression of CTGF-induced P-ERK1/2 in nuclei of breast cancer cells.

View larger version (57K): [in this window] [in a new window]   Fig. 5. S100A4 acts as a crucial downstream effecter of CTGF. (A) cDNA microarray analysis (Cy3-Cy5 merged image) showing the expression of S100A4, arrow indicates human S100A4. (B) Expression of S100A4 mRNA and protein by RT-PCR and western blotting, respectively. (C) Effects of ERK1/2 activation on CTGF-regulated S100A4 expression. MCF-7/neo and MCF-7/CTGF cells were treated with DMSO or PD98059 (20 µM) for 24 hours, and the levels of S100A4 were evaluated by western blotting. (D) MDA231/neo and MDA231/AS cells were transiently transfected by constitutively activated MEK1; 48 hours after transfection, expression levels of S100A4 were evaluated by western blotting. (E) Effects of S100A4 on CTGF-mediated cellular motility. MCF-7/neo and MCF-7/CTGF cells were stably transfected with pcDNA4 or pcDNA4-AS-S100A4, respectively (see Materials and Methods). Expression levels of S100A4 were detected by western blotting (upper panel). Cellular motility was measured by migration assay (lower panel). (F) MDA231/neo and MDA231/AS cells were stably transfected with pcDNA4 or pcDNA4-S100A4, respectively. Expression levels of S100A4 were detected by western blotting (upper panel). Cellular motility was measured by migration assay (lower panel). (G) S100A4 is involved in CTGF-mediated cytoskeletal changes. Cells were fixed in 3.7% paraformaldehyde and photographed under a light microscope (a,d,g,i). The fixed cells were co-stained with Texas-Red-phalloidin for F-actin (red; b,e,h,k) and monoclonal antibody against paxillin (green; c,f,i,l). (H) S100A4 expression is required for CTGF-mediated metastatic colonization. Lungs of female BALE/cAnN-Foxn1nu/CrlNarl nude mice were excised and photographed after the experimental metastasis assay.

View larger version (41K): [in this window] [in a new window]   Fig. 6. CTGF expression levels are correlated with S100A4 expression levels in samples of primary human breast cancer. (A) RT-PCR analysis of CTGF and S100A4 expression in nine representative samples of 24 analyzed primary breast cancer samples. N, non-tumor counterparts; T, tumor tissue. (B) Graph depicts the significant correlation between CTGF and S100A4 transcript levels determined in all 24 breast cancer samples by densitometric analysis and normalized to GAPDH control (r2=0.65, P=0.0012).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter