Published OnlineFirst November 11, 2010; DOI: 10.1158/1541-7786.MCR-10-0081 Molecular Cancer Angiogenesis, Metastasis, and the Cellular Microenvironment Research FMNL2 Enhances Invasion of Colorectal Carcinoma by Inducing Epithelial-Mesenchymal Transition 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. Ó2010 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 Ó2010 American Association for Cancer Research. acquisition of invasiveness and the possible signaling www.aacrjournals.org 1579 Downloaded from mcr.aacrjournals.org on October 2, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst November 11, 2010; DOI: 10.1158/1541-7786.MCR-10-0081 Li et al. 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 (À) 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 Â pathologists and examined for the presence of tumor cells. sample, and according to the equation: DDCt ¼ [Ct(target À À À 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 Â 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.