(CC Motif) Ligand 2 Mediates the Prometastatic Effect of Dysadherin

Research Article

Chemokine (C-C Motif) Ligand 2 Mediates the Prometastatic Effect of Dysadherin in Human Breast Cancer Cells

Jeong-Seok Nam,1 Mi-Jin Kang,1 Adam M. Suchar,1 Takeshi Shimamura,2 Ethan A. Kohn,1 Aleksandra M. Michalowska,1 V. Craig Jordan,3 Setsuo Hirohashi,4 and Lalage M. Wakefield1

1Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute; 2Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland; 3Fox Chase Cancer Center, Philadelphia, Pennsylvania; and 4Pathology Division, National Cancer Center Research Institute, Tokyo, Japan

Abstract of stratified squamous epithelium, show dysadherin expression (1). Dysadherin, a cancer-associated membrane glycoprotein, Collectively, the data suggest that overexpression of dysadherin down-regulates E-cadherin and promotes cancer metastasis. might contribute to tumor progression and could constitute a novel This study examined the role of dysadherin in breast cancer molecular target for the development of cancer therapeutics. progression. Expression of dysadherin was found to be highest In support of this hypothesis, it was shown that transfection of a in breast cancer cell lines and tumors that lacked the estrogen liver cancer cell line with the cDNA of dysadherin resulted in receptor (ER). Knockdown of dysadherin caused increased reduced cell-cell adhesiveness and down-regulation of E-cadherin protein (1). Furthermore, on injection into mouse spleens, association of E-cadherin with the actin cytoskeleton in breast cancer cell lines that expressed E-cadherin. However, knock- dysadherin transfectants formed a significantly larger number of down of dysadherin could still suppress cell invasiveness in intrahepatic metastatic nodules compared with the mock trans- cells that had no functional E-cadherin, suggesting the fectants, suggesting a capacity of dysadherin to promote metasta- existence of a novel mechanism of action. Global gene sis. Experimental overexpression of dysadherin in a pancreatic expression analysis identified chemokine (C-C motif) ligand cancer cell line also promoted metastasis in an orthotopic mouse 2 (CCL2) as the transcript most affected by dysadherin knock- model (5). Clinically, increased expression of dysadherin is down in MDA-MB-231 cells, and dysadherin was shown to significantly correlated with distant metastasis and poor prognosis regulate CCL2 expression in part through activation of the in human pancreatic, colorectal, thyroid, gastric, and tongue nuclear factor-KB pathway. The ability of dysadherin to cancers (6–10). Thus, both clinical and experimental data suggest that promote tumor cell invasion in vitro was dependent on the establishment of a CCL2 autocrine loop, and CCL2 secreted by dysadherin may play a particularly important role in cancer cell dysadherin-positive tumor cells also promoted endothelial cell invasion and metastasis, and that dysadherin expression could be migration in a paracrine fashion. Finally, experimental a useful biological predictor of tumor aggressiveness and poor suppression of CCL2 in MDA-MB-231 cells reduced their prognosis in human cancers (11). However, the molecular ability to metastasize in vivo. This study shows that mechanisms of dysadherin effects on cancer progression are still dysadherin has prometastatic effects that are independent of poorly understood. Because dysadherin expression was recently E-cadherin expression and that CCL2 could play an important shown to correlate with poor survival in a small cohort of breast role in mediating the prometastatic effect of dysadherin in cancer patients (1), here we have investigated further the possible functional involvement of dysadherin in breast cancer progression. ER-negative breast cancer. (Cancer Res 2006; 66(14): 7176-84) We find that dysadherin is particularly highly expressed in estrogen receptor (ER)–negative breast cancer and show that dysadherin Introduction may promote breast cancer metastasis by a novel E-cadherin- Dysadherin (also known as FXYD5, or Related to Ion Channel) independent mechanism that involves the up-regulation of chemo- was recently cloned as a cancer-associated membrane glycoprotein kine (C-C motif) ligand 2 (CCL2). (1). It is a member of the FXYD family of single-span type I membrane proteins, which interact with and modulate properties of Materials and Methods the Na+,K+ ATPase (2, 3). The dysadherin gene is up-regulated in cells transformed by several oncogenes, including E2a-Pbxl, v-Ras, Cell culture and reagents. The human breast cancer cell lines BT-474, Neu, and v-Src (4), and dysadherin is expressed to various extents in MCF-7, ZR-75B, T-47D, MDA-MB-468, SK-BR-3, MDA-MB-231, and Hs578T many different types of tumors, such as stomach, colon, pancreatic, and human umbilical cord vein endothelial cells (HUVEC) were obtained from American Type Culture Collection (Manassas, VA). MDA-MB-435 and and breast tumors (1). In contrast, only a limited number of normal MDA-MB-435LV/Br were kindly provided by Dr. Janet Price (University of cell types, including lymphocytes, endothelial cells, and basal cells Texas M. D. Anderson Cancer Center, Houston, TX). The human breast cancer cells were maintained in DMEM (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 Ag/mL streptomycin at 37jC in a humidified atmosphere Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). containing 5% CO2. HUVEC were cultured as described previously (12). Requests for reprints: Lalage M. Wakefield, Laboratory of Cell Regulation and MCF10A, MCF10AT1k, MCF10Ca1h, and MCF10Ca1a cells were kindly Carcinogenesis, National Cancer Institute, Building 41, Room C629, 41 Library Drive, provided by Dr. Fred Miller (Barbara Ann Karmanos Cancer Institute, MSC 5055, Bethesda, MD 20892-5055. Phone: 301-496-8351; Fax: 301-496-8395; E-mail: Detroit, MI), and cells were cultured as described previously (13). The [email protected]. I2006 American Association for Cancer Research. generation and culture of MDA-MB-231 clone (10A) and MDA-MB-231 doi:10.1158/0008-5472.CAN-06-0825 subline (S30) stably transfected with ER-a was described previously (14).

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InSolution nuclear factor-nB (NF-nB) activation inhibitor [6-amino-4- CA) using SYBR Green dye (Stratagene, Cedar Creek, TX). First-strand cDNA (4-phenoxyphenylethylamino)quinazoline] was purchased from Calbiochem was prepared from total RNA using a SuperScript III first-strand synthesis (La Jolla, CA). kit (Invitrogen). The quantitative RT-PCR was done in triplicates. Human Reverse transcription-PCR. Total RNA from human cancer cells was CCL2 (or dysadherin) mRNA levels were normalized with human 18S rRNA. isolated with the RNeasy Mini kit (Qiagen, Valencia, CA). Reverse The primer sets used in this study are as follows: CCL2 5¶-AAGATCT- transcription-PCR (RT-PCR) was then done using the SuperScript One-Step CAGTGCAGAGGCTCG-3¶ (forward) and 5¶-TTGCTTGTCCAGGTGGTCCAT- RT-PCR System (Invitrogen). The primer sets for amplification of human 3¶ (reverse), dysadherin 5¶-TCCCACTGATGACACCACGA-3¶ (forward) and glyceraldehyde-3-phosphate dehydrogenase were 5¶-AGGAAGAGAGAGAC- 5¶-AAAACCAGATGGCTTGAGGGT-3¶ (reverse), and 18S rRNA 5¶-CGCGGTT- CCTCACTGC-3¶ (forward) and 5¶-ATGACAAGGTGCGGCTCC-3¶ (reverse). CTATTTTGTTGGT-3¶ (forward) and 5¶-CCCTCTTAATCATGGCCTCA-3¶ The primer sets for amplification of human chemokine (C-C motif) receptor (reverse). 2 (CCR2) were purchased from Superarray (Frederick, MD). PCR products Stable knockdown of CCL2 in MDA-MB-231 cells. The retroviral-based were subjected to agarose gel electrophoresis and visualized by ethidium plasmid containing a short hairpin RNA (shRNA) to human CCL2 was bromide staining. purchased from Open Biosystems (Huntsville, AL). Nonsilencing shRNAs Immunoblotting. The cell lysates were subjected to 10% SDS-PAGE, and (Open Biosystems) were used as negative controls. Experimental procedures separated proteins in the gel were electroblotted to polyvinylidene for shRNA transfection were done according to the Open Biosystems difluoride membrane (Millipore, Canton, MA). Anti-dysadherin monoclonal technical manual. Stable transfectants were selected by incubation with antibody (mAb; NCC-M53, 1:500 dilution; ref. 6), E-cadherin mAb (1:500 puromycin (2 Ag/mL; Sigma-Aldrich), and knockdown of CCL2 expression dilution; ref. 15), ER-a mAb (1:250 dilution; Cell Signaling Technology, was determined by quantitative RT-PCR and ELISA analyses. Quantification Beverly, MA), and h-actin mAb (1:5,000 dilution; Sigma-Aldrich, St. Louis, of CCL2 secretion in cell culture supernatants was done by ELISA MO) were used as described previously (16). Triton X-100-soluble and (Quantikine Immunoassay; R&D Systems) according to the manufacturer’s Triton X-100-insoluble (cytoskeleton) fractions were prepared essentially as instructions. described previously (17). Promoter-reporter assay. After knockdown or overexpression of RNA interference. The small interfering RNA (siRNA) sequence chosen dysadherin was done as described above, cells in 12-well flat-bottomed to target dysadherin was at positions 141 to 162 in the nucleotide sequence plates were transfected with a total of 250 ng/well NF-nB-LUC promoter- of dysadherin (Genbank accession no. AB072911) as described previously reporter plasmid (a kind gift from Dr. Anita Roberts, National Cancer (5). Dysadherin siRNA was purchased from Dharmacon (Lafayette, CO). Institute, Bethesda, MD) together with 100 ng/well pRL-TK (Renilla) Nonspecific control I siRNA (Dharmacon) was used as a negative control. plasmid DNA for normalization using LipofectAMINE 2000 according to the siRNA transfection was done using Oligofectamine (Invitrogen) according manufacturer’s instructions. After transfection, cells were incubated for to the manufacturer’s instructions. For most experiments, cells were treated 16 to 18 hours, and cell lysates were harvested for assay using the Dual- for 48 hours with siRNA at a final concentration of 50 nmol/L. Dysadherin Luciferase Reporter Assay System (Promega, Madison, WI). Luminescence expression was determined by immunoblotting. was measured by VICTOR2 (Perkin-Elmer Life and Analytical Sciences, Transient transfection of dysadherin in T-47D cells. The expression Boston, MA). vector for dysadherin, pcDNA-L3HSV, has been previously described (1). Immunofluorescence. A total of 1 Â 105 cells per well were grown on Transfection was done with LipofectAMINE 2000 transfection reagent glass coverslips in 12-well flat-bottomed plates for 24 hours, and siRNA (Invitrogen) according to the manufacturer’s instructions. transfection was done as described above. The cells were fixed with 4% Matrigel invasion assay. Breast cancer cell invasion was assayed in 24- formaldehyde followed by 100% ethanol at À20jC. Permeabilization was well Biocoat Matrigel invasion chambers (8 Am; BD Biosciences Discovery done with 0.1% Triton X-100, and nonspecific binding was blocked with Labware, Bedford, MA) according to the manufacturer’s protocol. Briefly, 2% normal swine serum. Alexa Fluor 594–conjugated phalloidin (Molecular the top chamber was seeded with 1 Â 105 viable tumor cells in a serum-free Probes, Eugene, OR) was used to visualize F-actin. The samples were then medium (AIM-V medium; Invitrogen). The bottom chamber was filled with mounted with Vectashield (Vector Laboratories, Burlingame, CA) and DMEM supplemented with 10% FBS as a chemoattractant. CCL2-blocking examined by multiphoton fluorescence microscopy (Bio-Rad Laboratories). antibody (MAB679; R&D Systems, Minneapolis, MN) or isotype-matched Three visual fields per coverslip were randomly selected, avoiding any IgG antibody (MAB0041; R&D Systems) was added to the top chamber to a overlapping, for the morphologic analysis. The experiments were done in final concentration of 10 Ag/mL to block the CCL2 bioactivity. Recombinant triplicate. CCL2 protein (279-MC; R&D Systems) was added to the top chamber to a Endothelial cell migration assay. HUVEC chemotaxis was done using final concentration of 50 ng/mL. After 12 to 24 hours of incubation, micro-Boyden chambers as described previously (12). Briefly, polycarbonate migrated cells were stained with hematoxylin and counted in three different filters of 8 Am pore size (BD Biosciences Discovery Labware) were coated random fields using a light microscope at Â200. Invasion assays were done with fibronectin (10 Ag/mL; Sigma-Aldrich) overnight at 4jC. Conditioned in triplicate. medium from MDA-MB-231 cells transfected with control siRNA or Oligonucleotide microarray analysis. RNA was prepared from three dysadherin siRNA was placed in the lower compartment of the chamber, independent isolates of MDA-MB-231 cells transfected with dysadherin and 1% FBS/DMEM was used as the medium control. HUVEC (5 Â 105 siRNA or control siRNA using the RNeasy Mini kit according to the cells/mL) resuspended in 1% bovine serum albumin/DMEM were then manufacturer’s instructions (Qiagen). RNA quality and quantity was added to the upper compartment. Where indicated, CCL2 (15 ng/mL final assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). concentration) was added with conditioned medium in the lower The Affymetrix Gene Chip HG-133A (Affymetrix, Santa Clara, CA) was used compartment of the chamber. The chambers were incubated for 15 hours for analysis. cDNA synthesis and cRNA in vitro transcription, labeling, and at 37jC. After the filters were removed, the upper surface was scraped, fixed linear amplification were done using the Two-Cycle cDNA Synthesis kit and with methanol, and stained with Leukostat (Fisher Scientific, Pittsburgh, GeneChip IVT Labeling kit (Affymetrix). The in vitro transcription products PA). The number of cells was expressed as the number of migrated cells per were purified, fragmented, and hybridized to the oligonucleotide arrays as five fields at Â100 magnification. Each sample was tested in triplicate. recommended by the manufacturer. Raw data were processed with Robust In vivo experimental metastasis study. All animals were maintained Multiarray Average algorithm and quantile normalization to obtain gene according to the NIH Animal Care and Use Committee guidelines under summary measures (18). Differences in gene expression levels between the approved animal study protocols. To determine metastatic potential, two transfection groups were identified using univariate two-sample t test 7-week-old female severe combined immunodeficient (SCID) BALB/c mice (P < 0.001). The statistical computations were done using R and Affy (National Cancer Institute-Frederick, Frederick, MD) were injected i.v. with package of Bioconductor software project (http://www.bioconductor.org). 1 Â 106 cells in 0.1 mL PBS in the tail vein. Mice were euthanized and lungs Quantitative RT-PCR. Real-time quantitative RT-PCR was done in the were collected on day 42, inflated, and fixed with 10% buffered formalin. The iCycler iQ real-time PCR detection system (Bio-Rad Laboratories, Hercules, relative lung weight was calculated using the formula: lung weight / body www.aacrjournals.org 7177 Cancer Res 2006; 66: (14). July 15, 2006

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Cancer Research weight Â 100 (%). Macroscopic quantitation of metastases was done by human breast cell lines that represent different stages of breast counting the number of nodules on the surface of the lung. The presence of carcinogenesis. MCF10A is a spontaneously immortal human metastases was confirmed by pathologic examination of H&E-stained mammary epithelial cell line; MCF10AT1k is Ha-ras transfectant sections. of MCF10A that forms premalignant lesions on xenografting; In silico analysis of clinical microarray data. In silico analysis of MCF10Ca1h and MCF10Ca1a are tumorigenic variants of the published clinical microarray data was done using the database and analysis tools at http://www.oncomine.org. MCF10AT1k cells, forming well-differentiated, slow-growing tumors Statistical analysis. Unpaired parametric Student’s t test and nonpara- and poorly differentiated, rapidly growing tumors, respectively. metric Mann-Whitney U test were used to analyze the data unless otherwise Notably, only MCF10Ca1a can metastasize (13, 19). Immunoblot indicated in the text. analysis indicated that dysadherin protein was strongly increased in the high-grade metastatic cell line (MCF10Ca1a) and not in low- grade tumorigenic or premalignant lines (MCF10A, MCF10AT1k, Results and MCF10Ca1h). E-cadherin was also somewhat decreased in Dysadherin expression is elevated in more aggressive, MCF10Ca1a cells (Fig. 1A). In silico analysis of results from a large ER-negative breast cancer cell lines and tumors. To investigate clinical microarray study (20) also showed a significant trend the possible involvement of dysadherin in breast cancer progres- toward increased dysadherin mRNA expression with increasing sion, we examined dysadherin expression in a panel of related tumor grade in human breast cancers (Fig. 1B).

Figure 1. Expression of E-cadherin, dysadherin, and ER-a in human breast cancer cell lines and tumors. A, immunoblot analysis of E-cadherin and dysadherin in cell lines representing different stages in breast cancer progression. h-Actin was used as a loading control. B, dysadherin mRNA expression in human breast tumors as a function of tumor stage [extracted from van’t Veer et al. (20)]. C, immunoblot analysis of E-cadherin, dysadherin, and ER-a in human breast cancer cell lines. S30 is a subclone of MDA-MB-231 that has been stably transfected with ER-a. 10A is a control clone derived from the parental ER-negative MDA-MB-231 line. h-Actin was used as a loading control. D, dysadherin mRNA expression in human breast tumors as a function of ER status [extracted from Wang et al. (23) and van de Vijver et al. (24)].

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Figure 2. Effect of dysadherin knockdown on E-cadherin expression and invasion in vitro. A, immunoblot analysis of dysadherin in breast cancer cell lysates of parental cells (CON; Oligofectamine reagent alone) and cells transfected with nonspecific control I siRNA (NC)or dysadherin siRNA (siD). h-Actin was used as a loading control. B, immunoblot analysis of the expression and distribution of E-cadherin and dysadherin in lysates of breast cancer cells transfected with nonspecific control and dysadherin siRNA. Total, total protein extract; TS, Triton X-100-soluble fraction; TI, Triton X-100-insoluble fraction. C, effect of dysadherin knockdown on invasion of breast cancer cells through Matrigel. Columns, mean (n = 3 independent replicates); bars, SD. *, P < 0.001. D, immunoblot analysis of E-cadherin in the breast cancer cell lines used in the invasion assay.

ER-negative breast cancer is generally more aggressive and has a showed that knockdown of dysadherin causes an increase in poorer prognosis than ER-positive breast cancer (21, 22). We functional (Triton X-100-insoluble, cytoskeleton-associated) therefore examined the correlation between dysadherin and ER-a E-cadherin. These results suggest that dysadherin may not expression in breast cancer cell lines. Immunoblot analysis showed influence expression levels of total E-cadherin but rather may that expression of dysadherin protein is more prevalent in ER-a- influence interactions of E-cadherin with the actin cytoskeleton in negative than ER-a-positive breast cancer cell lines (Fig. 1C). breast cancer cell lines. In agreement with this finding, knockdown Moreover, in silico analysis of results from two large clinical of dysadherin did not cause any recovery of E-cadherin protein microarray studies (23, 24) showed that dysadherin mRNA levels expression in the E-cadherin-negative MDA-MB-231 cell line. correlated negatively with ER status in breast cancer (Fig. 1D). To understand the biological functions that dysadherin may Restoration of ER to the ER-negative cell line MDA-MB-231 affect during cancer progression, we examined whether knock- resulted in a reduction in dysadherin expression (Fig. 1C). Because down of dysadherin was able to suppress invasiveness of breast dysadherin has been shown previously to down-regulate cancer cells in vitro. Unexpectedly, we found that knockdown of E-cadherin (11), we also confirmed that E-cadherin expression dysadherin significantly reduced invasion through Matrigel in both was more prevalent in dysadherin-negative breast cancer cell lines E-cadherin-positive (MCF10Ca1a) and E-cadherin-negative cell than in dysadherin-positive cell lines (Fig. 1C). lines (MDA-MB-231 and MDA-MB-435LV/Br; Fig. 2C and D). These Dysadherin can promote invasion in vitro in the absence of results led us to hypothesize that dysadherin can promote cellular functional E-cadherin. To investigate further the functional role invasion by a novel mechanism that is independent of E-cadherin of dysadherin in breast cancer progression, we used siRNA expression. technology to knockdown dysadherin in breast cancer cells. Identification of CCL2 as a dysadherin target gene. To Expression of dysadherin protein was efficiently suppressed in investigate possible molecular mechanisms underlying the MCF10Ca1a, MDA-MB-231, and MDA-MB-435LV/Br cells trans- E-cadherin-independent effects of dysadherin, we compared gene fected with dysadherin siRNA, whereas the parent cells and cells expression patterns of MDA-MB-231 cells transfected with siRNA transfected with control siRNAs preserved dysadherin expression against dysadherin or nonspecific control siRNA. Seven hundred (Fig. 2A). twenty eight genes differed at the P < 0.001 level between To test whether dysadherin knockdown influences E-cadherin dysadherin siRNA-transfected and nonspecific control-transfected function in breast cancer cells, immunoblot analysis of E-cadherin cells. We filtered for genes that showed a >4-fold change in the expression and protein localization was done. Immunoblot analysis average difference between two groups, which gave a set of of total protein extracts showed that dysadherin siRNA did not 26 genes, 3 of which were up-regulated and 23 down-regulated by change total E-cadherin expression in E-cadherin-positive cell lines knockdown of dysadherin (Supplementary Table S1). (MCF10Ca1a and T-47D cells) compared with control counterparts The gene most highly affected by knockdown of dysadherin was (Fig. 2B). However, analysis of the distribution of E-cadherin CCL2. We focused on this gene, because chemokines have been www.aacrjournals.org 7179 Cancer Res 2006; 66: (14). July 15, 2006

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Cancer Research implicated previously in metastasis (25). First, we analyzed the plays an important role in dysadherin-driven biological responses, expression level of CCL2 mRNA by real-time quantitative RT-PCR we tested the invasiveness of breast cancer cells through Matrigel (Fig. 3A) and confirmed a >10-fold reduction of CCL2 mRNA level following experimental manipulation of CCL2 expression. Exoge- in MDA-MB-231 cells transfected with dysadherin siRNA compared nous addition of CCL2 had no effect on the basal invasiveness of with control counterpart. Using an ELISA assay, we then showed MDA-MB-231 cells. However, knockdown of dysadherin caused a that dysadherin siRNA caused a f7-fold decrease in the levels of f3-fold reduction in invasiveness of these cells (Fig. 4A), which CCL2 protein secreted into cell culture supernatants, confirming was largely restored by addition of recombinant CCL2 (Fig. 4A). that decreased mRNA expression results in reduced protein Moreover, the basal invasive activity of three breast cancer cells expression (Fig. 3A). Conversely, we investigated whether over- (MDA-MB231, MDA-MB-468, and Hs578T) was significantly expression of dysadherin could enhance CCL2 expression. T-47D inhibited by CCL2-blocking antibody (Fig. 4B). CCL2 therefore cells, which express relatively low levels of endogenous dysadherin, seems to be a positive autocrine regulator of invasion in multiple were transiently transfected with a dysadherin expression vector. breast cancer cell lines. The biological effects of CCL2 are primarily The dysadherin-overexpressing cells showed a f2-fold increase of mediated through binding to its receptor CCR2 (26, 27), and we CCL2 secretion compared with mock-transfected cells (Fig. 3B). confirmed that CCR2 mRNA was expressed in both T-47D and We next examined the correlation between dysadherin and CCL2 MDA-MB-231 cells (Supplementary Fig. S1). expression in additional breast cancer cell lines. Quantitative RT- The actin cytoskeleton is thought to be an important mediator of PCR analysis showed that expression of dysadherin mRNA was cell migration in the process of cancer cell invasion and metastasis generally associated with elevated CCL2 mRNA (Fig. 3C). These (28). It was shown previously that knockdown of dysadherin could data agree with those of Mestdagt et al. (30), who showed an increase transverse actin stress fibers, reduce cell motility, and inverse relationship between E-cadherin and CCL2 expression in a suppress the metastatic potential of human pancreatic cancer cells subset of these cell lines. As we have found with dysadherin, CCL2 (5). Using confocal microscopy, we found that MDA-MB-231 cells mRNA was also elevated in ER-negative compared with ER-positive transfected with dysadherin siRNA tended to have a larger, more human breast tumors in two large clinical microarray studies spread-out shape and increased number of transverse actin stress (Fig. 3D; refs. 23, 24). The data suggest that dysadherin is an fibers when compared with cells transfected with nonspecific important determinant of CCL2 expression in multiple breast control. Adding recombinant CCL2 to dysadherin siRNA-trans- cancer cell lines and clinical samples. fected cells decreased cell spreading and formation of filamentous Autocrine CCL2 mediates dysadherin effects on invasion transverse actin stress fibers (Fig. 4C). This result suggests that and actin organization in vitro. To determine whether CCL2 CCL2 expression may contribute to dysadherin-mediated invasion

Figure 3. Regulation of CCL2 by dysadherin. A, effect of dysadherin knockdown on expression of CCL2 mRNA and protein in MDA-MB-231 cells. The level of CCL2 mRNA was determined by quantitative RT-PCR and normalized with 18S rRNA in each sample; CCL2 protein secreted into the conditioned medium of transfected MDA-MB231 cells was detected by ELISA assay. B, CCL2 protein in conditioned medium from T-47D cells transiently transfected with empty vector (pcDNA3; EV) or dysadherin expression vector (L3HSV/pcDNA3; Dys). Top, secreted CCL2 protein determined by ELISA assay; bottom, immunoblot analysis of dysadherin. Columns, mean (n = 3); bars, SD. C, quantitative RT-PCR analysis of dysadherin (top) and CCL2 (bottom) mRNAs in human breast cancer cell lines normalized with 18S rRNA in each sample. Columns, mean of three determinations. D, CCL2 mRNA expression in human breast tumors as a function of ER status [extracted from Wang et al. (23) and van de Vijver et al. (24)].

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Figure 4. Involvement of CCL2 on tumor cell invasion, actin organization, and endothelial cell migration in vitro. A, invasion of MDA-MB-231 cells through Matrigel following transfection with nonspecific control or dysadherin siRNA. Where indicated, recombinant CCL2 protein (50 ng/mL) was added to the upper chamber. Columns, mean (n = 3 independent replicates); bars, SD. B, effect on Matrigel invasion assay of human breast cancer cells of blocking endogenous CCL2 activity by adding blocking antibody or control antibody (10 Ag/mL) to the upper chamber. Columns, mean (n = 3 independent replicates); bars, SD. C, F-actin staining (white)in MDA-MB-231 cells following transfection with nonspecific control or dysadherin siRNA. Recombinant CCL2 protein (50 ng/mL) was added to the cells for additional 24 hours after cells were first treated for 24 hours with siRNA. Bar, 20 Am. D, migration of HUVEC toward conditioned medium from MDA-MB-231 cells following transfection with nonspecific control or dysadherin siRNA. Where indicated, recombinant CCL2 protein (15 ng/mL) was added to the conditioned medium of dysadherin siRNA-transfected cells. Columns, mean (n = 3 independent replicates); bars, SD. *, P < 0.001. NS, no significance.

by causing reorganization of the actin cytoskeleton in an E- However, we did find that dysadherin overexpression significantly cadherin-independent fashion. up-regulated transcription from a NF-nB promoter-reporter Dysadherin could promote angiogenesis through paracrine construct in T-47D cells, whereas knockdown of dysadherin effects of CCL2 on endothelial cell migration. CCL2 has been significantly down-regulated transcription from the NF-nB pro- shown previously to act a direct mediator of angiogenesis (12). moter in MDA-MB-231 cells (Fig. 5A). Using quantitative RT-PCR, Given our results that dysadherin can regulate the level of CCL2 we showed that a NF-nB inhibitor significantly decreased CCL2 secreted by cancer cells, we tested whether breast cancer cell expression in MDA-MB-231 cells (Fig. 5B). The data suggest that conditioned medium could influence in vitro endothelial cell dysadherin enhances activation of the NF-nB pathway and that this migration. The migration of HUVEC induced by conditioned leads to increased CCL2 expression. medium from dysadherin siRNA-transfected MDA-MB-231 cells Knockdown of CCL2 significantly reduces invasion in vitro was 4-fold less than that induced by conditioned medium from the and metastasis in vivo. To determine whether CCL2 plays an control cells (Fig. 4D). Addition of CCL2 to the conditioned important role in cancer invasion and metastasis, we stably medium of dysadherin siRNA-transfected cells largely restored the knocked down CCL2 in MDA-MB-231 cells using shRNA (Supple- migration response, indicating that dysadherin-mediated control of mentary Fig. S2). To minimize the effect of clonal variation, we used CCL2 levels may influence angiogenesis by modulating endothelial two pooled populations of three CCL2 shRNA-transfected clones cell migration. (8, 9, 11) and three nonsilencing shRNA-transfected clones (1–3), Dysadherin up-regulates CCL2 expression in part through respectively. In vitro, the pool of CCL2 shRNA-transfected clones activation of the NF-KB signaling pathway. The CCL2 promoter showed a f5-fold decrease of CCL2 secretion compared with the contains sites for regulation by CAAT/enhancer-binding protein, control counterparts (Fig. 6A), which was comparable with the NF-nB, c-ETS, and h-catenin/TCF4 (29–31). We found no activation decrease seen following knockdown of dysadherin (Fig. 3A). of the h-catenin pathway in the MDA-MB-231 cells as indicated by In in vitro invasion assays, the pooled cells transfected with the absence of Top-Flash reporter activity (data not shown). CCL2 shRNA showed a f3-fold reduction in ability to invade www.aacrjournals.org 7181 Cancer Res 2006; 66: (14). July 15, 2006

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Cancer Research through Matrigel when compared with parent MDA-MB-231 cells highly expressed in ER-negative breast cancer cell lines and tumors, and the pool transfected with the nonsilencing shRNA (Fig. 6B); a subclass of breast tumors that has a generally poor prognosis. this is comparable with the reduction in invasion that was induced Unexpectedly, we found that dysadherin could promote invasion of by dysadherin siRNA (Fig. 4A). The decreased invasion is not due to breast cancer cells that did not express E-cadherin, suggesting a a decreased proliferation rate, as CCL2 shRNA and nonsilencing novel mechanism of action in this cell type. We identified the shRNA-transfected clone pools showed growth curves similar to chemokine CCL2 as a key mediator of dysadherin, with autocrine that of parental MDA-MB-231 (data not shown). stimulatory effects on tumor cell invasion and paracrine stimula- We then examined the effect of CCL2 knockdown in an tory effects on endothelial cell migration. The central importance experimental metastasis assay. We recognize that measurement of CCL2 was confirmed by the demonstration that CCL2 of lung colonization following i.v. injection of tumor cells does not knockdown significantly reduced metastasis of a dysadherin- recapitulate the complete spectrum of events that are required for positive breast cancer cell line in vivo. successful metastasis of a cell from a primary tumor. However, we Chemokines are emerging as important players in carcinogenic wished to be able to distinguish direct effects of CCL2 knockdown progression. Many tumors express one or more chemokines that specifically on steps involved in metastasis from effects that might may attract inflammatory cells, such as macrophages, which can be secondary consequences of CCL2 knockdown on primary tumor promote progression (25). Breast cancer cells have been shown growth or phenotype. previously to express the chemokine receptors CXCR4 and CCR7, Parent MDA-MB-231, the nonsilencing shRNA-transfected clone which increase the efficiency of tumor cell homing to metastatic pool, and the CCL2 shRNA-transfected clone pool were injected into target sites, such as the lung, where the cognate ligand CXCL12 the tail vein of SCID mice. The mice were euthanized 42 days after is highly expressed (32). Tumor-associated myoepithelial and injection of cells, and lung weight and metastases were examined. myofibroblast cells also overexpress CXCL12 and CXCL14, which The relative lung weight was significantly lower in the CCL2 shRNA promote proliferation, migration, and invasion of breast cancer group compared with control counterparts, suggesting a lower cells in a paracrine fashion (33). In some cases, a tumor cell may metastatic burden (Fig. 6C). Indeed, the number of metastatic lung express both chemokine and cognate receptor allowing establish- nodules in the mice injected with CCL2 shRNA-transfected clone ment of a tumor-promoting autocrine loop as has been shown for pool was significantly lower (f3-fold reduction) than those in the CCL5 (RANTES) in prostate cancer, CXCL8 [interleukin (IL)-8] in control counterparts (Fig. 6D). The presence of metastases was melanoma, and CXCL12 (SDF-1) in breast cancer (34–36). Here, we confirmed by pathologic examination (data not shown). Taken have shown that expression of dysadherin establishes a stimulatory together, these results clearly show that the knockdown of CCL2 by autocrine loop involving CCL2, which directly promotes invasion of stable shRNA reduced lung colonization in vivo following i.v. the tumor cell in vitro, without any effects on proliferation. To the injection of the breast cancer cells. Thus, CCL2 can increase the best of our knowledge, this is the first demonstration of an efficiency of one or more steps in the metastatic process that lie autocrine effect of CCL2 on a carcinoma cell, although an downstream of invasion of the primary tumor through the autocrine CCL2 loop has been implicated in the promotion of basement membrane and subsequent intravasation. Candidate transendothelial migration of myeloma cells (37). steps that might be affected would include tumor cell extravasation CCL2 (also known as MCAF or monocyte chemoattractant into the lung, and expansion of extravasated cells into macroscopi- protein-1) is a CC chemokine that is chemotactic for monocytes, cally visible colonies, dependent on successful angiogenesis. memory T cells, and natural killer cells (38). It is expressed by a wide variety of cancer types, and although CCL2 has been shown to display an antitumoral effect in some models (39, 40), a large Discussion number of studies showed that CCL2 generally facilitates tumor Metastasis is the most devastating consequence of cancer progression (41–43). Metastasis is a multistep process, and CCL2 progression, and much remains to be discovered about the could affect several of these steps, including chemoattraction of biological and molecular mechanisms that control the metastatic tumor-promoting leukocytes, tumor cell migration, and angiogen- process. Dysadherin has recently been implicated as a molecular esis (12, 44). Here, we have shown that the CCL2 that is secreted by player this process, and the prometastatic activity of dysadherin the tumor cell in response to dysadherin expression can promote has been attributed to its ability to down-regulate E-cadherin (11). tumor cell invasion in an autocrine manner and can also exert In the present study, we have investigated the role of dysadherin paracrine effects on endothelial cell recruitment that could enhance specifically in breast carcinogenesis. We showed that dysadherin is angiogenesis. Relevant to the autocrine invasion activity, CCL2

Figure 5. Effect of dysadherin expression status on NF-nB pathway activation. A, cells were transfected with NF-nB-LUC and Renilla plasmid DNA after overexpression or knockdown of dysadherin in T-47D and MDA-MB-231 cells, respectively. Cells were then incubated for an additional 18 hours before determination of luciferase activity. Luciferase activities were normalized based on Renilla expression. Columns, mean (n = 3); bars, SD. B, quantitative RT-PCR analysis of CCL2 mRNA in MDA-MB-231 cells after treatment for 24 hours with NF-nB inhibitor (1 Amol/L). The level of CCL2 mRNA was normalized with 18S rRNA in each sample. Columns, mean (n = 3); bars, SD.

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Figure 6. Effect of knockdown of CCL2 on invasion of MDA-MB-231 cells in vitro and lung metastasis in vivo. A, CCL2 protein expression determined by ELISA assay in supernatants from MDA-MB-231 parental cells and pooled clones transfected with CCL2 shRNA (shC)ora nonsilencing control shRNA (shS). Columns, mean (n = 3); bars, SD. B, invasion through Matrigel of parental MDA-MB-231 cells and pooled clones transfected with CCL2 shRNA or a nonsilencing control shRNA. Columns, mean (n = 3 independent replicates); bars, SD. C, relative lung weights of SCID mice at necropsy 42 days after tail vein injection with injected with MDA-MB-231 cells transfected with CCL2 shRNA or a nonsilencing control shRNA. D, total number of grossly visible metastatic nodules in the lung of individual mice injected with MDA-MB-231 cells as in (C). The median value is indicated for each group (parental MDA-MB-231 group, 8 mice; nonsilencing control shRNA group, 7 mice; CCL2 shRNA group, 8 mice).

seemed to mediate the effects of dysadherin on cytoskeletal is changed as a direct result of signaling from dysadherin or whether rearrangement. Our results support the previous finding that many of the changes are secondary to dysadherin-induced effects on dysadherin is able to modulate actin structures, stimulate cell cell shape. In rabbit synovial fibroblasts, cell shape change and motility, and contribute directly to the metastatic potential of reorganization of the actin cytoskeleton is associated with Rac human pancreatic cancer cells (5), and we identify a novel activation and the production of reactive oxygen species, leading to molecular mediator of this effect. Most importantly, we confirmed activation of the NF-nB pathway (49). Conceivably, dysadherin that CCL2 expression by the tumor is necessary for efficient effects on cell shape might initiate a similar cascade of events in metastasis of a dysadherin-expressing breast cancer cell line in vivo. breast cancer cells. Because the NF-nB inhibitor did not fully block Currently, little is known about signal transduction downstream the dysadherin-induced up-regulation of CCL2, dysadherin almost of dysadherin. In the present study, we showed for the first time certainly modulates other signal transduction pathways in addition that expression of dysadherin was associated with enhanced to NF-nB to bring about the observed gene expression changes. activity of the NF-nB pathway and further that NF-nB pathway Dysadherin is a member of the FXYD family that regulates Na+,K+ activation contributes substantially to the regulatory effect of ATPase (3, 50). The Na+,K+ ATPase has been shown to act as a signal dysadherin on CCL2. CCL2 expression has been shown previously transducer, in addition to being an ion pump (51, 52), so dysadherin to be controlled at the transcriptional level through a NF-nB- might also contribute to cancer metastasis through mechanisms mediated mechanism in other cell types (45–47). Our global gene involving changes in ion flux leading to effects on the protein kinase expression analysis showed that knockdown of dysadherin C and extracellular signal-regulated kinase 1/2 pathways. These enhanced the expression of NF-nB-repressing factor and reduced possibilities are under active investigation. the expression of several positive regulators of the NF-nB pathway In summary, we have shown that dysadherin may play an (MAP3K7IP2, SECTM1, and FKBP1A; data not shown), suggesting important role in ER-negative breast cancer by promoting invasion that dysadherin may affect this pathway at multiple levels. and metastasis through a mechanism that involves enhanced Although the molecular details remain to be elucidated, it is signaling through the NF-nB pathway, leading to increased nevertheless clear that dysadherin expression status can affect at production of the tumor-promoting chemokine, CCL2. Because least one signal transduction pathway that is highly relevant to antagonists of both the NF-nB pathway and of CCL2/CCR2 are tumorigenesis. Various inflammatory agents, carcinogens, tumor under active development for the treatment of chronic inflamma- promoters, and the tumor microenvironment can activate NF-nB, tory disorders (48, 53), these mechanistic insights suggest new and tumor invasion is regulated by numerous NF-nB-regulated approaches to the treatment of this particularly aggressive subclass gene products, including matrix metalloproteinases, urokinase-type of breast cancer. plasminogen activator, IL-8, and chemokines (31). Furthermore, NF-nB activity is constitutively high in ER-negative breast cancers Acknowledgments and cell lines and is elevated in breast with a low cancers with a Received 3/3/2006; revised 3/31/2006; accepted 5/9/2006. low ER content compared with those with high ER (reviewed in Grant support: Intramural Research Program of the NIH, National Cancer ref. 48), so it is tempting to speculate that dysadherin contributes Institute. The costs of publication of this article were defrayed in part by the payment of page significantly to this phenomenon. charges. This article must therefore be hereby marked advertisement in accordance Dysadherin knockdown altered the expression of several hundred with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Mario Anzano and Anthony Vieira for expert technical assistance with genes. Because rigorous kinetic experiments are challenging with the mice, Glenn Merlino for critical reading of the article and helpful discussions, and knockdown technology, it is not clear to what extent gene expression Janet Price for generously providing the MDA-MB-435 and MDA-MB-435LV/Br cells. www.aacrjournals.org 7183 Cancer Res 2006; 66: (14). July 15, 2006

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