FMNL2 Enhances Invasion of Colorectal Carcinoma by Inducing Epithelial-Mesenchymal Transition

Molecular Cancer Angiogenesis, Metastasis, and the Cellular Microenvironment Research

Yufa Li1,2, Xiling Zhu1,4, Yuanfeng Zeng1,3, Jianmei Wang1,3, Xiaojing Zhang1,3, Yan-qing Ding1,3, and Li Liang1,3

Abstract FMNL2 is a member of diaphanous-related formins that control actin-dependent processes such as cell motility and invasion. Its overexpression in metastatic cell lines and tissues of colorectal carcinoma has been associated with aggressive tumor development in our previous study. But its specific role in cancer is largely unknown. Here we report that FMNL2 is involved in epithelial-mesenchymal transition (EMT) maintenance in human colorectal carcinoma cells. A positive correlation between FMNL2 and vimentin expression and an inverse correlation between FMNL2 and E-cadherin expression were found in colorectal carcinoma cell lines and cancer tissues. Specific knockdown of FMNL2 led to an epithelial-state transition, confirmed by the cobblestone-like phenotype, upregulation of E-cadherin, a-catenin, and g-catenin, and downregulation of vimentin, snail, slug. Loss of FMNL2 expression lowered the ability of TGF-b to induce cell invasion and EMT, as shown by morphology and the expression levels. Upregulation of vimentin, slug, snail, downregulation of E-cadherin and activation of receptor-Smad3 phosphorylation were observed in M5 and MDCK cells induced by TGF-b, whereas altered expression of these markers was not obvious in FMNL2-depleting M5 cells. High levels of activation of p-MAPK þ and p-MEK, but not p-PI3K and p-AKT, were observed in SW480/FMNL2 cells compared with control cells. Treatment with U0126 could abrogate the activation of p-MAPK and p-MEK, whereas LY294002 treatment had no effect on the PI3K/AKT pathway. In conclusion, these findings identify a novel EMT and tumor promoting function for FMNL2, which is involved in TGF-b–induced EMT and colorectal carcinoma cell invasion via Smad3 effectors, or in collaboration with MAPK/MEK pathway. Mol Cancer Res; 8(12); 1579–90. 2010 AACR.

Introduction downregulation of epithelial differentiation markers, and transcriptional induction of mesenchymal markers, such as Epithelial-mesenchymal transition (EMT) is a morpho- vimentin and fibronectin. Transforming growth factor-b genetic process in which cells lose their epithelial character- (TGF-b) was first described as the inducer of EMT in istics such as cell polarity, cell-cell contact, and gain normal mammary epithelial cells (MEC; ref. 5) and now is mesenchymal properties such as increased motility (1). recognized as a master regulator of EMT in a variety of cell EMT was originally described during embryogenesis (2) types and tissues (6). in developmental processes such as gastrulation, renal orga- FMNL2 is a member of the diaphanous-related formins nogenesis, and the formation of neural crest (3). Accumu- (DRF) that are key regulators of the actin cytoskeleton and lating evidence has shown that EMT is reactivated in a act as effectors of Rho family guanosine triphosphatases variety of conditions including cancer progression and (GTPase;refs.7,8).Recently,DRFshaveemergedasa metastasis (4). At the molecular level, EMT is defined by diverse family of ubiquitous, highly conserved multido- the loss of cell-cell adhesion molecules (e.g., E-cadherin), main proteins involved in a growing range of cellular processes including filopodium formation, cell migration, cytokinesis, cell adhesion, and cell polarity (9, 10), which Authors' Affiliations: 1Department of Pathology, School of Basic Medical are frequently deregulated during pathologic situations Sciences, and 2Department of Pathology, Zhujiang Hospital, Southern such as tumor cell transformation and metastasis (11-13). Medical University, and 3Guangdong Province Key Laboratory of Mole- cular Tumor Pathology, Guangzhou, Guangdong Province, People’s Currently, some of the DRFs, such as mDia1, have been Republic of China and 4Department of Oncology, General Hospital of reported to be critically involved in cancer cell progression Armed Police Forces, Beijing, People’s Republic of China andinvasion(14,15).Butuntilnow,literatureon Corresponding Author: Yan-qing Ding and Li Liang, Department of FMNL2 has been scarce and its specific function in tumor Pathology, School of Basic Medical Sciences, Southern Medical Univer- sity, 1838, North Guangzhou Street, Baiyun Region, Guangzhou 510515, biology has not been elucidated (16-18). Our laboratory Guangdong Province, People’s Republic of China. E-mail: dyq@fimmu. was the first to identify FMNL2 as a metastasis associated com or [email protected]. Phone/Fax: 86-20-61642148 gene of colorectal cancer (CRC; ref. 19). In this study, we doi: 10.1158/1541-7786.MCR-10-0081 investigated the role of FMNL2 in EMT responsible for 2010 American Association for Cancer Research. acquisition of invasiveness and the possible signaling

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pathway involved in EMT during colorectal carcinoma was in accordance with a relatively simple and reprodu- progression. cible protocol (21). The stainingintensitywasscoredas0 (negative), 1 (weak), 2 (medium), and 3 (strong). Extent Materials and Methods ofstainingwasscoredas0(0%),1(1%to25%),2(26% to 50%), 3 (51% to 75%), and 4 (76% to 100%), Cell culture according to percentages of the positive staining areas Human CRC cell lines SW620, SW480, and HT29 in relation to the whole cancer area or entire section with differing metastatic abilities were obtained from for the normal samples. The sum of the intensity and American Type Culture Collection (ATCC). SW480/ extent score was used as the final staining score. Tumors M5 cell line is a subline of SW480 with enhanced ability having a final staining score of 3 or more were considered for hepatic metastasis developed after consecutive in vivo positive. The staining of FMNL2 was assessed as follows: selection at our laboratory (20). All cell lines were cultured () meant a final staining score of less than 3; (þ) a final in Dulbecco's modified Eagle's medium (GIBCO) with staining score of 3; (þþ)afinalstainingscoreof4;and 100 IU/mL penicillin, 100 mg/mL streptomycin, and 10% (þþþ) a final staining score of 5 or more. heat-inactivated fetal bovine serum (FBS) in humidified 5% CO2 atmosphere at 37 C. For TGF-b and inhibitors Real-time PCR assays treatment, 10 mmol/L MEK inhibitor U0126 or 10 Total RNA was extracted using Trizol reagent (Invitro- mmol/L PI3K inhibitor LY294002 (Cell Signal Technol- gen) and cDNA was synthesized by oligo dT primed reverse ogy) was added in the cultured cells every 2 days. The cells transcription from 2 mg of total RNA using an access RT were stimulated by 10 ng/mL human recombinant TGF- system (Promega). Reverse transcription was carried out for b1 (Peprotech) diluted with serum-free medium contain- 20 minutes at 42C. Real-time PCR was performed using ing 2 g/L bovine serum albumin for time periods of 12, Mx3000P Real-time PCR System (Stratagene) and SYBR 24, and 48 hours. PremixEx TaqTM (TaKaRa), using the following thermal cycling profile: predegeneration at 95C for 10 seconds, Patients and tissue specimens followed by 50 cycles of amplification (95C for 30 seconds, This study was conducted on a total of 168 paraffin- predicted TM value for 30 seconds, 72C for 30 seconds). embedded samples collected retrospectively from archival The dissociation curve analysis was used to validate the material stored at the Department of Pathology, Southern amplification of a single product. Each reaction consisted of Hospital (Guangzhou, China). Samples included 30 cases of 12.5 mL SYBR Premix ExTaq, 2 mL cDNA template, 0.5 normal colorectal mucosa, 93 cases of CRC tissues, 45 cases mL each primer set (10 mmol/L), and 9.5 mL nuclease-free of metastatic lymph nodes, and 10 cases of metastatic livers. water. Relative quantity of target transcripts in each sample All tissues obtained were reviewed by at least 2 experienced was expressed as n-fold differences relative to control, or 1 pathologists and examined for the presence of tumor cells. sample, and according to the equation: DDCt ¼ [Ct(target Pathologic diagnosis and classification were made based on gene) Ct(GAPDH)]experimental group [Ct(target gene) the system of the International Union Against Cancer. The Ct(GAPDH)]control group. Each sample was tested 3 times research protocol was approved by the Ethics Committee at and all other quantities were expressed as an n-fold differ- Nanfang Hospital, and consent was obtained from all ence relative to the corresponding control group. Gene- patients for the study. specific primers were as follows: FMNL2 (sense, 50-TGG CAT CGT GTA TCG AGG CTA A-30, 175bp); E-cad- Immunohistochemistry herin (sense, 50-TTA AAC TCC TGG CCT CAA GCA Sections were deparaffinized, rehydrated, and endogen- ATC-30, 139bp); vimentin (sense, 50-TGA GTA CCG 0 ous peroxidase was inhibited with 0.3% H2O2 methanol. GAG ACA GGT GCA G-3 , 119bp). For antigen retrieval, slides were boiled in 0.01 mol/L (pH 6.0) sodium citrate buffer for 15 minutes in a microwave Western blot oven. After blocking with 5% normal goat serum, primary Cells were washed twice with cold phosphate-buffered anti-FMNL2 monoclonal antibody (Abnova Corpora- saline (PBS) and lysed on ice in RIPA buffer (1 PBS, 1% tion), anti–E-cadherin (BD Corporation), and anti- NP40, 0.1% SDS, 5 mmol/L EDTA, 0.5% sodium deox- vimentin (Thermo Fisher Corporation) in blocking buffer ycholate and 1 mmol/L sodium orthovanadate) with pro- (1:50) were applied and the slides were incubated at 4C tease inhibitors and quantified by bicinchoninic acid overnight. Following incubation with biotinylated sec- method. Protein lysates (50 mg) were resolved on 6% ondary antisera, the streptavidin-biotin complex/horse- SDS polyacrylamide gel, electrotransferred to polyvinyli- radish peroxidase was applied. Finally, the visualization dene fluoride membranes (ImmobilonP; Millipore) and signal was developed with 3,30-diaminobenzidine tetrahy- blocked in 5% nonfat dry milk in Tris-buffered saline, drochloride, and slides were counterstained with hema- pH 7.5 (100 mmol/L NaCl, 50 mmol/L Tris, and 0.1% toxylin. The immunohistochemically stained tissue Tween-20). Membranes were immunoblotted overnight at sections were reviewed and scored separately by 2 pathol- 4C with anti-FMNL2 monoclonal antibody (Abnova ogists blinded to the clinical parameters. The scoring Corporation), anti–a-catenin and anti–b-catenin (BD Cor- approach used in the assessment of immunostaining poration); anti-slug and anti-snail (Abcam Corporation);

1580 Mol Cancer Res; 8(12) December 2010 Molecular Cancer Research FMNL2 Enhances Invasion by Epithelial Mesenchymal Transition

anti-MAPK, anti–p-MAPK, anti-MEK, anti–p-MEK, anti- In vitro invasion assay PI3K, anti–p-PI3K, anti-AKT, and anti–p-AKT (Cell Sig- In vitro invasive ability was tested by Boyden Chamber naling Technology, Inc.); anti-GAPDH antibody (Santa assay. Invasion Chamber has 8-mm pores in the polyethy- Cruz Biotechnology), followed by their respective horse- lene terephthalate (PET) membrane, which is coated with radish peroxidase-conjugated secondary antibodies. Signals matrigel (BD Biosciences). First, the invasion chambers were detected by enhanced chemiluminescence (Pierce). were rehydrated with RPMI 1640 (serum free) for 2 hours at 37 C in 50 mL/L CO2 atmosphere. RPMI 1640 with RNA interference 100 mL/L FBS was added to the lower compartment as the We designed 2 different siRNA duplexes of FMNL2 chemotactic factor. Then 1.5 105 tumor cells in serum- (GeneBank NM_001904): siRNA1 (sense, 50-GAT CCG free Dulbecco's Modified Eagle's Medium were added to CTG GAG GAG CGA TTT GCC ATT CAA GAC GTG the upper compartment of the chamber. Each cell group was GCA AAT CGC TCC TCC AGT TTT TTG TCG ACA- plated in 3 duplicate wells. After incubation for 48 hours, 30); siRNA2 (sense, 50-GAT CCG CCT ACC TCC TGA the noninvasive cells were removed with a cotton swab. CAA AGC CTT CAA GAC GGG CTT TGT CAG GAG Cells that had migrated through the membrane and stuck to GTA GGT TTT TTG TCG ACA-30). The 2 pairs of the lower surface of the membrane were fixed with metha- double-stranded DNA were cloned into plasmid vector nol and stained with hematoxylin. Finally, the cells in lower Pgenesil-1. DNA was purified with a Mini Plasmid Pur- compartment of the chamber that had invaded the lower ification Kit (Qiagen Co. Ltd.) and digested with appro- sides of the PET membrane were counted under a light priate restriction enzyme, SalI. Fragments were electro- microscope in 5 random visual fields (200). phoresed on 1% agarose to verify the insertion of sequences. For transfection, SW480/M5 and SW480 cell lines were Statistical analysis divided into negative control group and 2 test groups. Statistical analysis was carried out using SPSS 13.0 for Approximately 1 106 cells per well were plated in 6-well Windows. Results were presented as mean SD. Statistical plates in medium containing 10% FBS to grow overnight to differences of immunohistochemistry (IHC) were calcu- 90% confluency. All cells were transfected with 3 mgof lated using Kruskal-Wallis and Mann-Whitney test. The plasmids using Lipofectamine2000 according to instruc- independent samples t test was used for real-time RT-PCR. tions (Invitrogen). After 2 days, cells were selected for stable Statistical differences between groups were evaluated by 1- integrants by culturing in a complete medium containing way analysis of variance. Least-significant difference (LSD neomycin (800 ng/mL). Independent colonies resistant to test) was used for multiple comparisons. All P values for neomycin were picked up and subcultured in 14 days of multiple testing were corrected by Bonferroni correction. G418 selection. Western blot was performed to examine the P < 0.05 was considered statistically significant. knockdown of FMNL2 expression. Results Construction and transfection of FMNL2 recombinant plasmids Expression status of FMNL2, E-cadherin, and vimentin Because the coding region of FMNL2 is very long with in CRC cell lines and tissues the length of 3279bp, which makes transfection difficult, To determine the involvement of FMNL2 in EMT of we constructed the functional FMNL2-CT vector. Miralles CRC cell lines, we first examined the expressions of et al. (22) reported that the FH2 domain of Dia1 could FMNL2, EMT epithelial maker E-cadherin, and promote stress fiber formation and transcriptional activation mesenchymal maker vimentin in 4 human CRC cell lines of the MAL/SRF pathway through its actin-polymerizing with different metastatic abilities. Among the cells studied activity. The functional FH1-FH2-DAD (CT) domain of in Figure 1A, SW620 cells were originally established from FMNL2 was amplified by nested PCR and then inserted the lymph node metastasis in a patient with colon ade- into the pcDNA3/Flag (Invitrogen). Outer primers: for- nocarcinoma, whereas SW480 cells were derived from ward 50-CAT TGT CCA TGG GGT CAG AAG T-30, primary colon adenocarcinoma. SW480/M5 cells were reverse 50-GGC AGT TTC ATT CAC GCA CA-30; inner established by in vivo selection and had the high hepatic primers: forward 50-AAC GGT ACC CCC TTG CCC metastatic ability (20). Results of real-time PCR indicated CCT CCT CCA CC-30, reverse 50-CCA CTC GAG TCA that the expression of FMNL2 and vimentin in highly CAT TGT TAT TTC GGC ACC-30. The PCR conditions metastatic SW620 and SW480/M5 cell lines was higher were as follows: 95C for 3 minutes, followed by 30 cycles than in lowly metastatic SW480 and HT29 cells, whereas of amplification (94C for 30 seconds, 55C for 40 seconds E-cadherin was inversely expressed (P < 0.05; Fig. 1A). with outer primers or 68C for 40 seconds with inner Western blot analysis confirmed the similar changes in primers, 72C for 2 minutes). The correct coding regions of these cell lines (Fig. 1B). Results of cell IHC showed that all plasmids were confirmed by sequencing. Transient in the lowly metastatic SW480 and HT29 cell lines, transfection was performed using Lipofectamine (Invitro- FMNL2 and vimentin were expressed at low levels. But gen) as vehicle according to the manufacturer's instructions. highly metastatic SW620 and SW480/M5 cells had The plate was incubated for 48 hours until it was ready for increasing expression of vimentin and reduced expressions further assay. of E-cadherin (Fig. 1C).

www.aacrjournals.org Mol Cancer Res; 8(12) December 2010 1581 Li et al.

A 8 B 7 FMNL2 6 5 FMNL2 E-cadherin E-cad 4 vimentin 3 Vim 2

-fold related to sw480 1 GAPDH N 0 SW620 SW480/M5 SW480 HT29 SW620 SW480/M5 SW480 HT29

FMNL2

E-Cad

VIM

SW620 SW480/M5 SW480 HT29

Figure 1. Expression status of FMNL2, E-cadherin, and vimentin in colorectal carcinoma cell lines. A, real-time RT-PCR analyses of FMNL2, E-cadherin, and vimentin in highly metastatic SW620, SW480/M5 cells and less metastatic SW480, HT29 cells. The relative mRNA levels with the use of control SW480 were normalized to 1. Values were given as mean and SD from more than 3 independent experiments when compared with control cells. *, P < 0.05. B, Western blot analyses of FMNL2, E-cadherin, and vimentin in 4 cell lines. GAPDH was shown as a control. C, immunohistochemical analyses of FMNL2, E-cadherin, and vimentin in 4 cell lines.

To investigate the involvement of FMNL2 in EMT in expression of E-cadherin was decreased or even lost in vivo, we then examined the expression status of FMNL2, E- CRC tissues and lymphatic and hepatic metastases. E- cadherin, and vimentin in archived paraffin-embedded cadherin was expressed more highly in normal mucosa than human CRC tissues, normal tissues, metastatic lymph in CRC tissues (P < 0.01). But there was no significant nodes and livers. The expression of FMNL2 was gradually difference in E-cadherin expression between CRC tissues increased in normal mucosa, CRC tissues, metastatic lymph and metastatic lymph nodes or livers (P ¼ 0.187; Fig. 2B nodes and livers. The expression of FMNL2 in CRC tissues and C; Table 2). Vimentin was found dominantly in was higher than in normal mucosa (P < 0.01) and high levels stromal cells around the carcinoma cells. Occasionally, of FMNL2 expression were observed in metastatic lymph positive signal of vimentin was observed in CRC tissues nodes and livers compared with those in CRC tissues (P ¼ with poor differentiation and lymphatic and hepatic metas- 0.017; Fig. 2A, Table 1). Expression of E-cadherin was tases. The expression of vimentin in CRC tissues was higher mostly found in normal mucosa, whereas there seemed to be than in normal mucosa (P < 0.01). Higher levels of very low levels of FMNL2 in the control tissues. The vimentin in metastatic lymph nodes and livers were seen

1582 Mol Cancer Res; 8(12) December 2010 Molecular Cancer Research FMNL2 Enhances Invasion by Epithelial Mesenchymal Transition

AB CD

FMNL2

E-Cad

VIM

Figure 2. Expression status of FMNL2, E-cadherin, and vimentin in colorectal carcinoma tissues by immunohistochemistry. Serial sections of normal mucosa (A), colorectal carcinoma tissues (B), metastatic lymph nodes (C), and metastatic livers (D). The expression of FMNL2 was gradually increased in normal mucosa, CRC tissues, metastatic lymph nodes, or livers. Expression of E-cadherin was mostly found in normal mucosa, and decreased or even lost in colorectal carcinoma tissues and lymphatic or hepatic metastases. Vimentin was found dominantly in stromal cells around the carcinoma cells. Occasionally, positive signal of vimentin was observed in colorectal carcinoma tissues with poor differentiation and lymphatic or hepatic metastases. (3,30- Diaminobenzidine, 200)

Table 1. Expressions of FMNL2 in normal mucosa, CRC tissues, and metastatic lymph nodes and livers

Group þ þþ þþþ Total

Normal 22 8 0 0 30a,b CRC 93a,c,d Well differentiated 6 24 20 2 52e Poorly differentiated 10 23 7 1 41e LN metastasis 10 7 22 6 45b,c Hepatic metastasis 0 3 4 3 10d

aZ ¼5.700, P ¼ 0.000. bZ ¼5.243, P ¼ 0.000 (Mann-Whitney Test); c2 ¼ 48.559, V ¼ 4, P ¼ 0.000 (Kruskal-Wallis Test). cZ ¼2.390, P ¼ 0.017. dZ ¼3.230, P ¼ 0.026. eZ ¼2.455, P ¼ 0.014.

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Table 2. Expressions of E-cadherin in normal mucosa, CRC tissues, and metastatic lymph node and livers

Group þþþþþþTotal

Normal 0 0 0 30 30a,b CRC 93a,c,d Well differentiated 0 3 27 22 52e Poorly differentiated 5 14 20 2 41e LN metastasis 8 12 15 10 45b,c Hepatic metastasis 4 4 2 0 10d

aZ ¼6.585, P ¼ 0.000. bZ ¼6.213, P ¼ 0.043 (Mann-Whitney Test); c2 ¼ 146.243, V ¼ 4, P ¼ 0.000 (Kruskal-Wallis Test). cZ ¼2.019, P ¼ 0.043. dZ ¼5.875, P ¼ 0.031. eZ ¼5.320, P ¼ 0.000.

than in CRC tissues (P ¼ 0.037; Fig. 2D; Table 3). Two- blestone-like phenotype, whereas SW480/M5 and variance of Pearson correlation analysis showed that the SW480 cells, maintained their spindle shaped and fibro- expression of FMNL2 was negatively correlated with that of blastic appearance in monolayer culture. We observed E-cadherin (r ¼0.338, P ¼ 0.000), but positively frequent appearance of multiple clusters of adherent cells correlated with that of vimentin (r ¼ 0.349, P ¼ 0.000). under FMNL2 knockdown. The morphologic changes The above results suggest that enhanced expression of imply that the FMNL2-depleting cells have undergone FMNL2 is at least partly correlated with EMT in CRC transdifferentiation from mesenchymal cells to epithelial cell lines and tissues. cells (MET). Then we detected the expressions of MET markers Requirement of FMNL2 for maintaining the following FMNL2 silencing. E-cadherin was upregulated mesenchymal state of CRC cells in both FMNL2-depleting cells compared with mock cells, To determine whether FMNL2 plays a contributory whereas vimentin was downregulated in FMNL2-depleting role in CRC invasion by inducing EMT, we knocked cells by real-time RT-PCR (P < 0.05), suggesting a switch to downFMNL2using2specificsiRNAs.Westernblot the epithelial state under FMNL2 knockdown (Fig. 3C and analysis confirmed that FMNL2 were depleted by the D). Western blot analysis further showed that the expres- specific knockdown in SW480/M5 and SW480 cells sion of E-cadherin was increased in FMNL2-depleting M5 (Fig.3A).Celldeath,whichwasassessedbytrypanblue and SW480 cells, whereas that of vimentin was decreased. staining, did not increase in these knockdown cells Another 2 EMT epithelial markers a-catenin and g-catenin (Fig. 3B). We observed that SW480/M5 and SW480 were also found to be increasingly expressed in FMNL2- cells developed morphologic changes under the FMNL2 depleting M5 and SW480 cells. In addition, FMNL2 knockdown (Fig. 4A). As shown by phase contrast image, knockdown caused decrease of 2 EMT-stimulating tran- the FMNL2-depleting CRC cells appeared with a cob- scription factors Snail and Slug in FMNL2-depleting cells

Table 3. Expressions of vimentin in normal mucosa, CRC tissues, and metastatic lymph nodes and livers

Group þ þþ þþþ Total

Normal 30 0 0 0 30a,b CRC 93a,c Well differentiated 29 13 10 0 52d Poorly differentiated 6 19 13 3 41d LN metastasis 7 22 14 2 45b,c Hepatic metastasis 3 5 2 0 10e

aZ ¼5.630, P ¼ 0.000. bZ ¼6.693, P ¼ 0.000 (Mann-Whitney Test); c2 ¼ 59.896, V ¼ 4, P ¼ 0.000 (Kruskal-Wallis Test). cZ ¼2.086, P ¼ 0.037. dZ ¼3.792, P ¼ 0.000. eZ ¼4.382, P ¼ 0.017.

1584 Mol Cancer Res; 8(12) December 2010 Molecular Cancer Research FMNL2 Enhances Invasion by Epithelial Mesenchymal Transition

100% ABP > 0.05 P > 0.05 75% Negative FMNL2-siRNA1control FMNL2-siRNA1Negative controlFMNL2-siRNA1FMNL2-siRNA1

FMNL2 50%

GAPDH Cell viability 25%

SW480/M5 SW480 0% C M5/mock SW480/mockM5/siRNA1 SW480/siRNA1 5 * * 4

3 480/FMNL2-siRNA1 E-cadherin E M5 M5/FMNL2-siRNA1M5/FMNL2-siRNA2SW480 SW SW480/FMNL2-siRNA2 Vimentin 2 * * Vim 1 Folds related to M5/mock 0 E-cad M5/mock M5/FMNL2-siRNA1 M5/FMNL2-siRNA2 D α-catenin 5 * * γ-catenin 4

3 snail E-cadherin Vimentin 2 slug 1 actin

Folds related to SW480/mock 0 SW480/mock SW480/FMNL2-siRNA1 SW480/FMNL2-siRNA2

Figure 3. Requirement of FMNL2 for maintaining the mesenchymal state of colorectal carcinoma cells. A, knockdown of FMNL2 by 2 specific siRNAs by Western blot in SW480/M5 and SW480 cell lines. GAPDH was shown as a control. B, cell viability of FMNL2-depleting cells by trypan blue staining on day 5 after distinct siRNA transfection. C, D, real-time RT-PCR analyses of E-cadherin and vimentin in FMNL2-depleting SW480/M5 and SW480 cells. The relative mRNA levels with the use of control mock cells were normalized to 1. Values were given as mean and SD from more than 3 independent experiments when compared with control cells. *, P < 0.05. E, Western blot analyses of vimentin, E-cadherin, a-catenin, b-catenin, snail, and slug in FMNL2-depleting SW480/M5 and SW480 cells compared with those in mock cells. Actin was shown as a control.

(Fig. 3E). These data suggest that FMNL2 is actively at the time of 24 or 48 hours, the cultured M5/FMNL2 involved in maintaining EMT in CRC cells. cells penetrating the artificial basement membrane (28.34 8.72 or 47.44 5.42) were less than in M5 (83.02 FMNL2 is involved in TGF-b–induced EMT and CRC 5.739 or 100.12 8.865; P < 0.01; Fig. 4B). TGF-b cell invasion stimulation can enhance invasive abilities of both cells Multiple signal transduction pathways have been identi- (Fig. 4B). There was a 2.5-fold induction of tumor cell fied to be involved in the induction of EMT. Previous invasion in M5/FMNL2 cells and an approximate 6-fold reports showed that TGF-b signaling promotes the metas- induction in M5 cells by TGF-b (Fig. 4C). It indicates that tasizing and invasive properties of some cancers, presumably repression of endogenous FMNL2 reduces CRC cell by inducing EMT of the cells (23). TGF-b elicits EMT and invasion and strongly diminishes the stimulatory effects in vivo metastasis via Smads and complementary non-Smad of TGF-b. effectors, such as Rho GTPases and p38 MAPK (24). In Furthermore, TGF-b treatment for a period of 48 hours CRC, it was reported that TGF-b itself is overexpressed induced an EMT in the canine kidney derived epithelial cell (25). To elucidate the relationship between FMNL2 and line (MDCK) and M5 and SW480 cells, as illustrated by the TGF-b signaling in CRC cells, we analyzed the effect of acquisition of a fibroblast-like phenotype. MDCK is com- recombinant TGF-b on SW480/M5, SW480, and their monly used in EMT study and can undergo typical mor- corresponding FMNL2-depleting cells. First, we examined phologic changes of EMT induced by TGF-b (26). MDCK the potential of TGF-b to stimulate M5 cell invasion by cells appeared spindle-shaped and fibroblastic-like instead of Boyden chamber assays. Invasion through the ECM is an the cobblestone-like phenotype induced by TGF-b. important step in tumor metastasis. The results showed that FMNL2-depleting cells represented mixed fibroblast-like

www.aacrjournals.org Mol Cancer Res; 8(12) December 2010 1585 Li et al.

TGF-β (−)

TGF-β (+) MDCK M5/mock M5/FMNL2-siRNA1 SW480/mock SW480/FMNL2-siRNA1 B TGFβ1(+) 12 h TGFβ1(+) 24 h TGFβ1(+) 48 h

180 * 160 140 * 120 100 SW480/M5/mock 80 TGFβ1(+) 12 h TGFβ1(+) 24 h TGFβ1(+) 48 h 60 40

Number of invaded cells 20 0 h +) 12 h +) 24 h +) 48 h +) 12 +) 24 h +) 48 h β1( β1( β1( β1( β1( β1( TGF TGF TGF TGF TGF TGF

SW480/M5/FMNL2/siRNA1 SW480/M5/mock SW480/M5/FMNL2/siRNA1

Figure 4. FMNL2 is involved in TGF-b–induced cell invasion of colorectal carcinoma. A, epithelial-like changes induced by FMNL2 knockdown and mesenchymal-like changes induced by TGF-b. Colorectal carcinoma cell lines included SW480/M5 and SW480. MDCK cells were used as a positive control of mesenchymal-like changes induced by TGF-b. Phase contrast studies were on day 6 after the siRNA transfection. B, cell invasion induced by TGF-b at the time periods of 12, 24, and 48 hours in FMNL2-depleting cells and mock cells. The data were represented with SW480/M5 cells. The cells were placed in a 3-dimensional Matrigel invasion chamber and cells that invaded through Matrigel were fixed, stained, and counted. C, number of cells that invaded through the Matrigel was counted in 10 fields under 20 objective lens. *, P < 0.05.

and epithelial cells, and obviously had less fibroblast-like tion whereas the expression levels of Smad3 was not affected cells than mock cells (Fig. 4A). Loss of FMNL2 expression by TGF-b treatment, even after 48 hours postapplication lowered the ability of TGF-b to induce a spindle-like cell (Fig. 5D). However, the expression changes of these mar- shape. kers were not obvious in M5/FMNL2 cells as compared Second, we detected the altered expressions of EMT with those in M5 and MDCK cells (Fig. 5D). Reduced markers induced by TGF-b. At the mRNA level, TGF- cancer cell migration and invasion were associated with b–induced EMT over a period of 24 or 48 hours was reduced EMT, and loss of FMNL2 expression lowered the reflected by upregulation of vimentin and downregulation ability of TGF-b to induce EMT, as shown on the mor- of E-cadherin in M5 and MDCK cells compared with those phologic and the expression levels. This evidence makes it without TGF-b stimulation (P < 0.05; Fig. 5A and C). reasonable to presume that FMNL2 is involved in TGF- TGF-b stimulation resulted in a dramatic increase of b–induced mesencymal state in CRC cells. FMNL2 expression in M5 cells (P < 0.05; Fig. 5A). In contrast, the FMNL2-depleting M5 cells showed no Ras-MAPK pathway is involved in FMNL2-induced obvious expression changes of EMT under treatment of EMT TGF-b (P > 0.05; Fig. 5B). At the protein level, there was In our previous study, the gene expression profile low expression of E-cadherin and high expression of vimen- between FMNL2-depleting M5 cells and M5 cells revealed tin, transcription factor snail, and slug in M5 and MDCK significant downregulation of PI3K, AKT, MAPK, and cells on TGF-b stimulation. These cells also showed rapid MEK genes in FMNL2-depleting cells. Analysis of the and sustained activation of receptor–Smad3 phosphoryla- signaling network on the chip data indicated that PI3K/

1586 Mol Cancer Res; 8(12) December 2010 Molecular Cancer Research FMNL2 Enhances Invasion by Epithelial Mesenchymal Transition

AB SW480/M5 SW480/M5/siRNA1 3.5 4 3.5 3 * 3 2.5 * 2.5 * E-cadherin 2 E-cadherin 2 Vimentin Vimentin 1.5 1.5 FMNL2 FMNL2 1 1 0.5 -folds related to M5/0 h

N 0.5 *

Folds related to M5/siRNA1/0 h 0 M5/siRNA1/0 h M5/siRNA1/ M5/siRNA1/ 0 β β M5/0 h M5/TGF-β/24 h M5/TGF-β/48 h TGF- /24 h TGF- /48 h

24 h β/ β/48 h h h h GF- /24 /48 0 β -β β/24 h β/48 h h /0 TGF- CDGF- iRNA1/ MDCK M5/0 h M5/T M5/TGF- M5/s M5/siRNA1/TM5/siRNA1/TGF-MDCK MDCK/ MDCK/TGF 4.5 Vim 4 * 3.5 E-cad 3 * 2.5 E-cadherin p-Smad3 2 Vimentin FMNL2 Smad3 1.5

1 snail Folds related to MDCK/0 h 0.5 * 0 slug MDCK/0 h MDCK/TGF-β/24 h MDCK/TGF-β/48 h

GAPDH

Figure 5. FMNL2 is involved in TGF-b–induced EMT of colorectal carcinoma. Real-time RT-PCR analyses of FMNL2, E-cadherin, and vimentin in TGF-b–induced EMT at the time periods of 24 and 48 hours. The data were represented with SW480/M5 (A), FMNL2-depleting M5 (B), and MDCK cells (C). The relative mRNA levels with the use of control cells were normalized to 1. Values were given as mean and SD from more than 3 independent experiments when compared with control cells. *, P < 0.05. D, Western blot analyses of vimentin, E-cadherin, p-Smad3, Smad3, snail, and slug in TGF-b induced EMT at the time periods of 24 and 48 hours. SW480/M5, FMNL2-depleting M5, and MDCK cells were used. GAPDH was shown as a control.

AKT and MAPK/MEK signaling pathways may be involved p-MAPK and p-MEK and the expression changes of EMT in the downstream regulation of FMNL2 (data not shown). marker vimentin and E-cadherin, whereas LY294002 treat- To test the involvement of the 2 pathways in EMT induced ment had no effect on the PI3K/AKT pathway (Fig. 6B). by FMNL2, we prepared exogenous FMNL2-overexpres- The results suggest that Ras-MAPK pathway is involved in sing SW480 cells, in which FMNL2 expression was at low FMNL2-induced EMT. level, and then treated them with MEK inhibitor U0126 or PI3K inhibitor LY294002 for 2 days. Compared to SW480/ Discussion mock cells, the expression of FMNL2 mRNA was higher in þ SW480/FMNL2 cells, but lower in FMNL2-depleting FMNL2 is a member of the DRFs that are key regulators SW480 cells as compared by real-time RT-PCR (P < of the actin cytoskeleton exerted through their forming 0.05; Fig. 6A). Western blot analysis showed that high levels homology (FH)2 domains and act as effectors of Rho family of activations of p-MAPK and p-MEK, but not p-PI3K and GTPases (15). Recently, DRFs have been shown to be þ p-AKT, were observed in SW480/FMNL2 cells compared involved in essential cellular processes such as cytokinesis, with control cells, with no change in the amount of total cell movement, and polarity, which are frequently deregu- protein. Knockdown of FMNL2 gene in SW480 cells lated during pathologic situations such as tumor cell trans- resulted in the reduced expressions of the 4 phosphorylated formation and metastasis (27). Three members of the DRF proteins, whereas the expression levels of total protein were family (DRF1–DRF3) are reported to be required for not affected. We also detected upregulation of vimentin and invadopodia formation and invasion of breast tumor cells þ downregulation of E-cadherin in SW480/FMNL2 cells (28). FMNL2 was identified as a candidate metastasis compared with control cells, whereas FMNL2 knockdown associated gene in our previous study (19). However, the showed the opposite effects (Fig. 6B). Treatment of SW480/ precise role of FMNL2 in mediating tumor cell invasion and þ FMNL2 cells with U0126 could abrogate the activation of metastasis has not been investigated.

www.aacrjournals.org Mol Cancer Res; 8(12) December 2010 1587 Li et al.

b-catenin relocalization. As defined by Hay (29), EMT A results in the complex loss of epithelial traits (including loss 3.0 of E-cadherin, plakoglobin, and cytokeratins, and the dis- mantling of adherens junctions and desmosomes), accom-

2.5 panied by the total acquisition of mesenchymal * characteristics, including expression of vimentin, N-cad-

2.0 herin, fibronectin, and an elongated ``fibroblast-like'' morphology with capacity to execute invasive motility (30). E- cadherin expression is irreversibly lost in invasive lobular 1.5 breast cancer (31). The cytoplasmic/nuclear relocalization

SW480/mock of b-catenin from the adherens and tight junctions is a 1.0 common process in the EMT associated with tumor invasion (32). This transition is considered to be an important -folds FMNL2 expression related to

N 0.5 event during malignant tumor progression and metastasis * (33, 34). 0.0 To determine the involvement of FMNL2 with EMT of SW480/mock SW480/flag- SW480/-FMNL2- CRC, we first detected the expressions of FMNL2, E- FMNL2-CT siRNA1 cadherin, and vimentin in 4 metastatic CRC cell lines

4002 and CRC tissues. In highly metastatic SW620 and NA1 0126 ′ siR ′/LY29 ′/U SW480/M5 cells, FMNL2 and vimentin were expressed B 2 2 L2 MNL2- MN at higher levels, whereas E-cadherin was inversely expressed. 80/F W480 S SW480/FMNLSW4 SW480/FMNLSW480/F The expression of FMNL2 was gradually increased in normal mucosa, CRC tissues, metastatic lymph nodes, P-MAPK and livers. Two-variance of Pearson correlation analysis showed that the expression of FMNL2 in normal mucosa, MAPK CRC tissues, and lymph node metastases was negatively correlated with those of E-cadherin and CK, whereas P-MEK positively correlated with that of vimentin. These observa- tions suggest that enhanced expression of FMNL2 is at least MEK partly correlated with EMT in CRC cell lines and tissues. Then we showed that FMNL2 conferred EMT to CRC P-PI3K epithelial cells, with gain of biological features consistent with neoplastic transformation and invasiveness. Knock- PI3K down of FMNL2 in SW480/M5 cells could induce the conversion of spindle-shape mesenchymal morphology to P-AKT cobblestone-like epithelial morphology. Consistent with the morphologic change, E-cadherin, a-catenin, and g-catenin AKT were increased whereas vimentin was reduced or even lost during the transition. Transcriptional factors such as snail Vim (35), slug (36), and Twist (37) were unveiled as key regulators in induction of EMT. We discovered that E-cad FMNL2 knockdown also inhibited the expression of snail

GAPDH and slug. Several growth factors such as TGF-b, hepatocyte growth factor (HGF), and matrix metalloproteinase (MMP-3) have been reported to induce EMT and to be associated with a Figure 6. Involvement of Ras-MAPK pathway in FMNL2-induced EMT. A, real-time RT-PCR analysis of FMNL2 in SW480/FMNL2þ and more invasive phenotype in pancreatic cancer (38-40). SW480/FMNL2 cells. The relative mRNA levels with the use of control TGF-b was first described as inducer of EMT in normal mock cells were normalized to 1. Values were given as mean and SD MECs (5) and is now recognized as a master regulator of from more than 3 independent experiments when compared with EMT in a variety of cell types and tissues (6). In various control cells. *, P < 0.05. B, E-cadherin, vimentin, PI3K, AKT, MAPK, tumor cell line, TGF-b signaling induces snail, slug, and MEK, and their phosphorylated proteins were determined by Western blot in SW480/FMNL2þ and SW480/FMNL2 cells. SW480/FMNL2þ SIP1, which may then proceed to repress the expression of cells were also treated with MEK inhibitor U0126 and PI3K inhibitor E-cadherin (35). LY294002. GAPDH was shown as a control. Our study reveals that FMNL2, in association with the TGF-b signaling pathway, is required for maintenance of EMT is characterized by the disassembly of cell-cell invasive ability and the mesenchymal state in CRC cells. contacts, reorganization of the actin cytoskleleton, and TGF-b stimulation can enhance the invasive abilities of cell–cell separation by reduced E-cadherin expression and SW480/M5 and M5/FMNL2 cells. Repression of

1588 Mol Cancer Res; 8(12) December 2010 Molecular Cancer Research FMNL2 Enhances Invasion by Epithelial Mesenchymal Transition

FMNL2 reduced M5 cell invasion under unstimulated total protein. Upregulation of vimentin and downregulation þ conditions and strongly diminished the stimulatory effects of E-cadherin were also seen in SW480/FMNL2 cells, of TGF-b. Reduced cancer cell migration and invasion were whereas FMNL2 knockdown showed the opposite effects. associated with reduced EMT. TGF-b treatment for a Treatment with U0126 could abrogate the activation of p- period over 24 and 48 hours induced an EMT changes MAPK and p-MEK, and the expression changes of EMT in MDCK and SW480/M5 cells, as illustrated by the marker vimentin and E-cadherin, whereas LY294002 treat- acquisition of a fibroblast-like phenotype, downregulation ment had no effect on the PI3K/AKT pathway. The results of E-cadherin, upregulation of vimentin, and activation of suggest that Ras-MAPK pathway is involved in FMNL2- receptor–Smad3 phosphorylation. But the expression induced EMT. changes of these markers were not obvious in M5/FMNL2 Taken together, our studies indicate that (1) the cells compared with those in M5 and MDCK cells. Loss of enhanced expression of FMNL2 is at least partly correlated FMNL2 expression lowered the ability of TGF-b to induce with EMT in CRC cell lines and tissues; (2) FMNL2 is EMT, which suggests that FMNL2 contributes to the required for maintaining the mesenchymal state of CRC acquisition of a mesenchymal and highly migrating pheno- cells; (3) FMNL2 is involved in TGF-b–induced EMT and type in CRC cells induced by TGF-b. CRC cell invasion via Smad3 effectors; (4) Ras–MAPK The transduction pathway involved in EMT is very pathway is also involved in FMNL2-induced EMT. These complex. Tyrosine kinase receptors get activated and med- findings identify a novel EMT and tumor promoting iate downstream signaling after binding to various ligands function for FMNL2 in metastasis of colorectal carcinoma. such as EGF and TGF-b. EMT occurs by hyperactivation of 1 pathway or, more probably, by simultaneous activation Disclosure of Potential Conflicts of Interest of more pathways, which either leads to Snail-mediated downregulation of the E-cadherin gene, as Ras–Raf–MEK– No potential conflicts of interest were disclosed. MAPK, PI3K–Akt, TGF-b–Smads, and Wnt–b-catenin Acknowledgment signaling do, or directly affects cell adhesion and/or the cytoskeletal dynamics, as accomplished by Src, TGF- We thank Professor Geevan for editing the English writing. b–Par6–Smurf1, and Rho GTPases. Thereby, activation of a single molecular pathway can lead to 1 or more features Grant Support of EMT (41). PI3K/Akt-dependent signaling pathways served to regulate hypoxia-induced EMT of hepatocellular carcinoma cells (42). Also well documented was the fact that This work was supported by the National Natural Science Foundation of China (no. 30870945, 30770977), the Research Fund for the Doctoral Program of Higher the Ras-RAF-MAPK pathway played an indispensable role Education of China (no. 2009443312009, 20094433110001), Natural Science in EMT induced by activation of receptor tyrosine kinase of Foundation of Guangdong Province (no. 5200512), National Basic Research Program growth factors such as HGF, VEGF, EGF, and bFGF (18, of China (973 Program, No.2010CB529403), and Innovative Research Team Foun- dation in University (No. IRT0731). 43). Finally, we determined the signaling pathways that The costs of publication of this article were defrayed in part by the payment of page FMNL2 may participate in EMT of CRC according to our charges. This article must therefore be hereby marked advertisement in accordance previous cDNA chip data. Only activation of p-MAPK and with 18 U.S.C. Section 1734 solely to indicate this fact. p-MEK, but not p-PI3K and p-AKT, were observed in þ Received 03/16/2010; revised 10/19/2010; accepted 10/20/2010; SW480/FMNL2 cells, with no change in the amount of published OnlineFirst 11/11/2010.

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