Destrin Contributes to Lung Adenocarcinoma Progression By

Published OnlineFirst September 2, 2020; DOI: 10.1158/1541-7786.MCR-20-0187

MOLECULAR CANCER RESEARCH | CANCER GENES AND NETWORKS

Destrin Contributes to Lung Adenocarcinoma Progression by Activating Wnt/b-Catenin Signaling Pathway A C Hui-Juan Zhang1, Wen-Jing Chang1, Cai-Yun Jia1, Ling Qiao1, Jing Zhou1, Qing Chen1, Xiao-Wei Zheng1,4, Jian-Hua Zhang4, Hong-Chao Li5, Zheng-Yan Yang1, Zhong-Hua Liu3, Guang-Chao Liu1, Shao-Ping Ji2, and Feng Lu1

ABSTRACT ◥ Lung cancer, especially lung adenocarcinoma, is one of the with the poor prognosis of lung adenocarcinoma patients. Fur- most common neoplasms worldwide. However, the mechanisms thermore, we also found that DSTN promotes cell proliferation, underlying its initiation, development, and metastasis are still invasion, and migration in vitro, and facilitates subcutaneous poorly understood. Destrin (DSTN) is a member of ADF/cofilin tumor formation and lung metastasis via intravenous injection family. Its detailed biological function remains unknown, in vivo. Mechanically, DSTN associates with and facilitates although it is reported that DSTN is involved in cytoskeleton nuclear translocation of b-catenin, which promotes epithelial- remodeling and regulation of actin filament turnover. Recent to-mesenchymal transition (EMT). Taken together, our results evidence has shown that high expression of cofilin-1 is associated indicated that DSTN enhances lung cancer malignancy through with invasion and poor prognosis of several types of human facilitating b-catenin nuclear translocation and inducing EMT. tumors, but the detailed mechanism is still entirely unclear, Combined with multivariate analyses, DSTN might potentially particularly in lung cancer tumorigenesis and malignancy. Here, serve as a therapeutic target and an independent prognostic we report that DSTN was highly expressed in a mouse lung marker of lung adenocarcinoma. cancer model induced by urethane and in clinical lung adenocarcinoma tissue samples. Its expression level was positively Implications: This finding indicates that DSTN facilitates correlated with cancer development, as well as metastasis to the b-catenin nuclear translocation and promotes malignancy in lung liver and lymph nodes. Consistently, it was directly associated adenocarcinoma.

Introduction which results in the loss of opportunity for curative surgical resection and a poor prognosis (3). Lung adenocarcinoma is the most prevalent Lung cancer is the leading cause of cancer-related deaths in China histologic type of lung cancer, which accounts for approximately 40% and worldwide. Despite a great progress in cancer research and of all lung cancers. During the last few years, lung adenocarcinoma has therapeutic strategies over the last decades, the prognosis of lung an increasing frequency, and its prognosis has not been obviously cancer remains poor with a 5-year survival rate of less than 15% (1, 2). improved (4). The prognosis of lung adenocarcinoma is highly asso- Therefore, both understanding the mechanism of cancer development ciated with lymph node and distant metastasis. Therefore, it is of great and progression and early diagnosis are critical to lung cancer treat- interest to identify novel biomarkers that serve for early diagnosis and ment. Unfortunately, because of the lack of early and reliable diag- accurate prognosis prediction, which allow improving the prognosis. nostic biomarkers together with the limited understanding of its Proteomic technologies have been widely used in the global analysis carcinogenic mechanisms, more than 70% of the patients are found for lung cancer biomarker discovery (5). Although many proteomic in advanced stages accompanied by extensive invasion and metastasis, studies on lung adenocarcinoma have been reported (6, 7), to our knowledge, little is known about the changes in protein expressional proﬁles at the early-stage of human lung adenocarcinoma, neither any 1 Joint National Laboratory for Antibody Drug Engineering, Henan University, clinically established biomarkers available for early detection of lung 2 Kaifeng, P.R. China. Department of Biochemistry and Molecular Biology, adenocarcinoma. Comparative proteomics analysis of successive Medical School, Henan University, Kaifeng, P.R. China. 3Laboratory for NanoMedical Photonics, School of Basic Medical Science, Henan University, stages of human lung adenocarcinoma is the most direct and persua- Kaifeng, P.R. China. 4Department of Clinical Laboratory, Puyang Hospital of sive way to identify biomarkers for early diagnosis of lung adenocar- Traditional Chinese Medicine, Puyang, P.R. China. 5Department of Pathology, cinoma. However, a major obstacle in the analysis of tissue specimens Puyang Oilfeld General Hospital, Puyang, P.R. China. is tissue heterogeneity, in which different cells are collected. Further- Note: Supplementary data for this article are available at Molecular Cancer more, it is difﬁcult to obtain a large amount of early-stage clinical lung Research Online (http://mcr.aacrjournals.org/). adenocarcinoma tissue. H.-J. Zhang and W. -J. Chang contributed equally to this article. To search for the early biomarkers and explore the underlying mechanisms of initiation and metastasis of lung adenocarcinoma, in Corresponding Authors: Feng Lu, Henan University, Kaifeng 475004, P.R. China. Phone/Fax: 8637-1238-80398; E-mail: [email protected]; and this study, we developed a urethane-induced mouse lung adenocar- Shao-Ping Ji, [email protected] cinoma model, which exhibits similar histologic appearance and molecular changes to human lung adenocarcinoma (8, 9). We used Mol Cancer Res 2020;XX:XX–XX a laser capture microdissection (LCM) approach to purify the target doi: 10.1158/1541-7786.MCR-20-0187 cells from lung tissues of a mouse model with early carcinogenesis 2020 American Association for Cancer Research. lesion, then investigated the differential proteins in tumor and control

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tissue using isobaric tagging for relative and absolute protein quan- lungs were prepared for histologic, iTRAQ, and Western blotting tification (iTRAQ) combined with two-dimensional 2D-LC/MS-MS analyses. Five mice in each group were examined at each time point. approach. Subsequently, we further validated our observations by IHC and Western blot analysis. Among the identified 191 potential pro- Sample preparation and LC-ESI-MS/MS analysis teins, destrin (DSTN) protein expression (Dstn, tr|Q4FK36|) displayed Lung tissue protein sample preparation, iTRAQ labeling, LC- a significant upregulation (3.67-fold to control) in early-stage mouse electrospray (ESI)-MS/MS analysis and protein identification were lung cancer tissues compared with the control group. performed as described previously (18, 19). Additional details are DSTN belongs to the ADF/cofilin family, consisting of DSTN, provided in the Supplementary Data. cofilin-1, and muscle-specificcofilin-2 (10). These proteins are abun- dant and essential in almost all eukaryotic cell types. They involve in Clinical lung adenocarcinoma and adjacent nontumor lung cytoskeleton remodeling and precise regulation of the actin filament tissues turnover and take part in numerous cellular processes such as cell A total of 30 paired samples of lung adenocarcinoma and adjacent cytokinesis, proliferation, and membrane trafficking (11, 12). Recent nontumor lung tissues were obtained from patients who underwent studies showed that actin dynamics and their regulation of target surgery between 2013 and 2015 and preserved in the Department of proteins are not only essential for the healthy development and Pathology, Puyang Oilfield General Hospital (Henan, P.R. China). All function of the cells, but also play a crucial role in human diseases, patients gave written informed consent before sample collection. including cancers. However, most research on ADF/cofilin family None of the lung adenocarcinoma patients received radio- or chemo- proteins in metastatic invasion has focused on cofilin-1 (13, 14). As a therapy before surgery. Lung adenocarcinoma tissue microarray member of the ADF/cofilin protein family, the DSTN with the least (HLugA180Su02) purchased from Shanghai Outdo Biotech Co., Ltd. study, and is only found to regulate the migration and invasion of colon included 86 lung adenocarcinoma patients undergoing surgery cancer through regulating action dynamics (15). Its high expression between 2004 and 2009. This study was approved by the Ethic was the association with growth and perineural invasion of pancreatic Committee of the Medical School, Henan University (Kaifeng, P.R. cancer (16). However, its precise roles in human cancers, particularly China) and all methods in this study were carried out in accordance in lung adenocarcinoma, and its downstream signaling pathway in with the recommended guidelines. these processes, remain largely unclear. Here, we have found that overexpressed DSTN seems to be a Cell culture, expression plasmids, and stable transfection of functional oncogenic molecule at lung adenocarcinoma both shRNA in vitro and in vivo. For the first time, we have demonstrated that The human NSCLC cell lines, A549, HCI-H1299, HCI-H460, HCI- DSTN expression is associated with progression of lung cancer H1373, HCI-H1573, and immortalized lung epithelial cell line BEAS- development and liver metastasis in urethane-induced lung adeno- 2B were obtained from the Cell Bank of Type Culture Collection of the carcinoma model. IHC and Western blot analysis showed that the high Chinese Academy of Sciences (Shanghai, China). The cells were expression level of DSTN is closely related to lymph node metastasis routinely screened to confirm Mycoplasma-negative status, and cell and aggravates the clinical progression of human lung adenocarcino- line was authenticated by examination of morphology, growth char- ma. The knockdown of DSTN inhibits cell proliferation, migration, acteristics, and short tandem repeat analysis. There were enough and invasion, leading to an arrest of the cell cycle, and in turn promotes frozen vials for each cell line to ensure that all cell-based experiments apoptosis of NSCLC cells. Performing the rescue experiment by were carried out in the cells under test and cultured for 8 weeks or less. lentiviral overexpression of DSTN in knockdown cells, efficiently The cells were routinely cultured in RPMI1640 medium (Corning) restored cell proliferation and invasion and colleagues Furthermore, supplemented with 10% FBS (PAN Biotech), 100 U/mL penicillin, and we observed that overexpressed DSTN could upregulate the activity of 100 mg/mL streptomycin in a humidified cell incubator with an the Wnt/b-catenin signaling pathway by promoting nuclear translo- atmosphere of 5% CO2 at 37 C (all cells are cultured in the same calization of b-catenin and interaction of b-catenin with TCF4. These condition). Empty vector pcDNA3.1(þ), pCDH-CMV-MCS-EF1- observations provide new insights into the molecular function of Neo, and pCDH-CMV-MCS-EF1-puro were from YouBio. The DSTN as well as its aggravating mechanisms in the development and detailed information of cell culture and plasmids construction is given progression of lung adenocarcinoma. in the Supplementary Materials and Methods.

RNA extraction and qRT-PCR analysis Materials and Methods Total RNA was extracted from frozen tissues or cell lines using Reagent and antibodies TRIzol reagent (Takara) according to the manufacturer’s instructions. Cell culture media, reagent, and antibody information are presented For reverse transcription, 1 mg of total RNA was used with Reverse in the Supplementary Data. Transcriptase Kit (RR036A, Takara). Real-time PCR was performed using the TB Green Premix Ex Taq (RR420A, Takara) and ABI Prism Urethane-induced murine lung adenocarcinoma model 7900 System. The expression levels of genes were calculated using the C Six-week-old female BALB/c mice (Weitong Lihua Animal Co.) 2 DD t method, and GAPDH was used as endogenous control (20). were housed under pathogen-free conditions and all procedures The qRT-PCR primers used in this study were presented in Supple- involving mice followed Henan University Animal Care and Use mentary Table S6. Committee guideline. The urethane-treated group mice received an intraperitoneal injection of urethane (800 mg/kg body weight; Sigma) Rescue experiment dissolved in sterile 0.9% NaCl saline once a week for 12 consecutive To recover the expression of DSTN-silenced cells, A549 or H1299 weeks. Control group mice received parallel intraperitoneal injection cells with DSTN knockdown by sh1DSTN targeting 30UTR of DSTN of saline (17). Animals were sacriﬁced at 16, 20, 24, 28, and 32 weeks were transduced with lentiviral DSTN-overexpressing vector that was after the ﬁrst urethane injection with age-matched controls, and their bearing the resistance to Neo. Cells then were selected for 7 days with

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G418, then allowed to recover for 5 days, seeded, and allowed to grow DSTN aggravates tumor progression and liver metastasis in for 5 days. urethane-induced a mouse model of lung tumor IHC was performed using antibodies against DSTN (ab186754) to Coimmunoprecipitation assay measure DSTN expression levels. The results demonstrated that DSTN The total Cell protein was generated by lysing cells with RIPA buffer protein expression increased in a stepwise manner following the for 1 hour at 4C. The nuclear extraction was prepared using a NE-PER progression of lung lesion (Fig. 1A–E). Furthermore, the increasing Nuclear Cytoplasmic Extraction Reagent Kit (Pierce) according to the DSTN protein expression in the mouse model’s lung tumor tissues was manufacturer’s instructions. Coimmunoprecipitation (co-IP) assay also confirmed by Western blot analysis (Fig. 1F). Noticeably, at the was then performed as described previously (21, 22). Protein lysates end of the experiment (32th week posttreatment of urethane), con- were incubated with 5 mg of indicated primary antibodies overnight on comitant liver metastases of tumors were observed in 60% (7/10) of the a rotator at 4C, followed by adding 50 mL of equilibrated Protein A/G mice treated with urethane while no metastasis (0/10) was detected in beads (Santa Cruz Biotechnology) and incubation for another 3 hours the matched control group (Fig. 1G). To further evaluate whether on a rotator at 4C. The agarose beads were collected by centrifuging, DSTN upregulation was associated with tumor metastasis at tumor late followed by washing with lysis buffer to remove all of the nonspecif- stage, Western blot analysis was performed. As demonstrated ically bounded proteins. The bounded immune complexes were eluted in Fig. 1H, DSTN protein expression level was much higher in lung from the beads with corresponding elution buffer (elution is not tumor tissues of a mouse model with liver metastasis than mice without necessary), boiled with sample-loading buffer, and detected by West- liver metastasis. These results suggested that DSTN was involved in the ern blotting analysis. progression and metastasis of lung adenocarcinoma in the lung cancer model induced by urethane. Additional methods IHC, immunoblotting, immunofluorescence, Luciferase reporter DSTN was frequently upregulated in human lung gene assay, in vivo tumorigenesis and metastasis assay, and cell adenocarcinoma tissues and associated with poor prognosis of adhesion assay were performed using standard techniques. Invasion lung adenocarcinoma ability of cells was estimated by Matrigel invasion, wound-healing To determine the expression level of DSTN in human lung ade- assay, and soft agar assay. Proliferation was measured by colony nocarcinoma, we first examined the DSTN expression pattern in 20 formation and MTT assay. The flow cytometry analysis was used to pairs of human lung adenocarcinoma tissues and their adjacent assess cell-cycle distribution and levels of cell apoptosis. Additional nontumor tissues by Western blot analysis. As shown in Fig. 2A, details are included in Supplementary Data. DSTN levels were noticeable higher in the tumor tissues (T) than in the matched adjacent nontumor tissues (N; P < 0.05). Real-time PCR Statistical analysis assays also observed that the mRNA levels of DSTN were highly All the statistical data are presented as the mean SD of at least expressed in 55.2% tumor tissues (11/20; Fig. 2B). In addition, we also three independent experiments performed in triplicate. The signif- further examined the association of DSTN protein levels with lymph icance of the difference between groups was assessed with a two- node metastasis and different clinical TNM stages of lung adenocar- tailed t test or x2 analysis. Kaplan–Meier survival analysis and the cinoma using Western blotting analysis. As shown in Fig. 2C, the log-rank nonparametric test were used to evaluate the overall expression of DSTN protein levels in patients with lung adenocarci- survival of patients with lung adenocarcinoma concerning DSTN noma with lymph node metastasis was markedly higher than the expression. The effect of clinical variables on lung adenocarcinoma patient with no lymph node metastasis (P < 0.05). Moreover, with the patient survival was analyzed using Cox proportional hazards progression of clinical stages, DSTN expression increased in a stepwise regression analysis. P < 0.05 was considered statistically significant. manner evidenced by Western blotting analysis (Fig. 2D). Next, to We did all calculations and analyses with SPSS statistical software further assess the protein levels of DSTN in lung adenocarcinoma (version 20.0). tissues, IHC staining of DSTN was performed using the lung adenocarcinoma tissue microarray, which containing 86 human lung adenocarcinoma specimens and their adjacent nontumor tissues with Results complete clinicopathologic characteristics and follow-up data. As Identification of differentially expressed proteins in mouse lung shown in Fig. 2E, DSTN was detected in the cytoplasm and nucleus tumor model of tumor cells and was strongly expressed in 55.8% (46/86) tumor We first generated a mouse lung tumor model induced by tissues, whereas 88.4% (76/86) adjacent nontumor tissues showed no urethane (Supplementary Fig. S1A–S1F). These histologic indica- or low DSTN expression and only 11.6% (10/86) samples exhibited tors of tumor progression were consistent with studies reported by high DSTN expression. Finally, to characterize the roles of DSTN in others (23, 24). We found 191 differently expressed proteins by human lung adenocarcinoma development and progression, the rela- iTRAQ analysis (Supplementary Table S1). To verify the results of tionships between the DSTN protein expression and clinicopathologic mass spectrometry, we selected several differentially expressed parameters of patients with lung adenocarcinoma were analyzed. We proteins, such as Prdx5, Ctsd, Ctsh, Ctnnal, Jup, and DSTN, etc., found that high expression of DSTN in lung adenocarcinoma was for IHC and Western blot analysis (Supplementary Table S2). significantly correlated with late clinical stages (P ¼ 0.008) and with Representative experimental results were presented in Supplemen- lymph node metastasis (P ¼ 0.009). No significant correlations were tary Figs. S2 and S3. Moreover, we observed that destrin (Dstn, observed between DSTN expression and other clinicopathologic char- tr|Q4FK36|) expression was markedly high compared with the acteristics, such as gender, age, tumor size, and pathologic stages control group (3.67-fold; Supplementary Fig. S1G), and its link to (Supplementary Table S3). lung adenocarcinoma had not previously been reported. We further In addition, Kaplan–Meier analysis showed that patients with lung focus on assessing its potential as a biomarker for lung adenocar- adenocarcinoma with high DSTN expression exhibited poorer survival cinomas and related molecular mechanisms. than those with low expression (Fig. 2F). Moreover, we further

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Control 20W

A 100 mmB 100 mm

CD100 mm 100 mm 28W 32W EF 6.0 40 P = 0.041 P = 5.55e-8 5.0 35 16W 20W 24W 28W 32W P 30 = 1.10e-5 N T N T N T N T N T 4.0 25 DSTN P = 0.017 20 3.0 P = 0.002 P = 0.001 15 GAPDH 2.0 DSTN/ﬁeld 10 5 1.0 % of Cells expressing % of Cells 0 DSTN Relative expression Relative DSTN 0.0 Control 20W 28W 32W NC 16W 20W 24W 28W 32W

Normal NometastasisMetastasis

DSTN

Coﬁlin1

GAPDH Normal liver Liver metastasis

DSTN Coﬁlin1 8 7 6 5 4 3 2 indicated protein indicated 1 Relative expression of expression Relative 0

Normal Metastasis Nometastasis

Figure 1. Characterization of DSNT expression in lungs following urethane treatment. A–D, Representative images of IHC staining for DSTN in the lungs of control mice (A)and the mice at weeks 20 (B), 28 (C), and 32 (D) after urethane treatment. E, The number of cells expressing DSTN was evaluated by IHC in serial sections from lung tissues. F, The protein expression of DSTN in the tumor (T) and matched control (N) lung tissue extracts at the different time points (16W, 20W, 24W, 28W, 32W) were analyzed by Western blotting; the right panel is the quantified results by grayscale scanning (n ¼ 5). The equal amounts of protein samples were resolved on SDS- PAGE and detected with antibodies against DSTN. GAPDH was used as a loading control. G, 60% (7/10) of mice treated with urethane showed liver metastases 32 weeks posttreatment, H&E staining analysis in liver metastases and control liver tissues was performed. The blue arrows indicate clusters of tumor cells that have colonized in the lung. Scale bar, 100 mm. H, The protein expression of DSTN and cofilin1 in the lung of mice with liver metastases and without liver metastases at week 32 after urethane treatment and control were analyzed by Western blotting. Bottom, the quantified results by grayscale scanning (, P < 0.05; , P < 0.01).

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A B 2 5 6 9 11 12 16 20 Sample 16 N T NTNTNTN TNT N TNTN T DSTN 14 12 GAPDH 10 2.0 8

1.5 6 4 1.0 2 0.5

Relative mRNA expression of DSTN mRNA expression Relative 0 0.0 987654321 1110 12 19181716151413 20

DSTN Relative expression Relative DSTN TN C Lymph node 6.0 metastasis Nometastasis Metastasis Yes TNTNTNTN TNTNTNTN 4.0 No DSTN 2.0 GAPDH

expression level expression 0.0 Adjacent Tumor Relative protein of DSTN protein Relative D TNM I TNM II TNTNTN TNTNTN 8.0 TNM I DSTN TNM II GAPDH 6.0 TNM III TNM IV TNM III TNM IV 4.0 TNTNTN TNTNTN DSTN 2.0 expression level expression GAPDH 0.0

Relative protein of DSTN protein Relative Adjacent Tumor E

200 mm 200 mm200 mm 200 mm

FGP Lo, stage I + II vs. Hi, stage I + II. P = 0.006 Low DSTN vs. High DSTN, = 6.314e-5 Lo, stage III + IV vs. Hi, stage III + IV. P = 0.072 1.0 DSTN 1.0 Low DSTN 0.8 High 0.8 Lo, stage I + II Low-censored Hi, stage I + II 0.6 High-censored 0.6 Lo, stage III + IV 0.4 0.4 Hi, stage III + IV Lo, stage I + II-censored Cum survival 0.2 Cum survival 0.2 Hi, stage I + II-censored Lo, stage III + IV-censored 0.0 0.0 Hi, stage III + IV-censored 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Survival time (months) Survival time (months)

Figure 2. DSTN protein expression in the lung adenocarcinoma and Kaplan–Meier survival curves of overall survival with regards to DSTN expression in lung adenocarcinoma. A, Representative Western blotting analysis of DSNT protein expression levels in lung adenocarcinoma tissue samples (T) and matched adjacent nontumor tissues (N). GAPDH was used as loading control in the immunoblotting assay. Top, the signal intensity of DSTN and GAPDH; bottom, the quantiﬁed results by grayscale scanning. n ¼ 20; , P < 0.05. B, Real-time PCR analysis of DSTN mRNA levels in 20 paired lung adenocarcinoma tissues (T) and adjacent nontumor tissues (N). The expression of GADPH was used as loading control in real-time PCR assays. , P < 0.01; , P < 0.001. (Continued on the following page.)

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stratified analysis and found that patients with lung adenocarcinoma at A549 cells significantly increased tumor growth. As evidenced by a the clinic early stage with high DSTN survived significantly shorter significant increase in the average size and weight of tumors. Consis- than those with low DSTN expression (P ¼ 0.006; Fig. 2G). Cox tent with this observation, Ki-67 proliferation assays showed that to multivariate analysis showed that lymph node metastasis (HR ¼ 2.850, compare with control tumors, overexpression of DSTN tumor cells P ¼ 0.021) and DSTN high expression (HR ¼ 2.149, P ¼ 0.007) were showed an increased cell proliferation rate. In contrast, cell prolifer- negatively correlated with postoperative survival and positively cor- ation rate was significantly inhibited by DSTN knockdown (Supple- related with mortality. While DSTN low expression favored the mentary Fig. S5). These data together suggest that DSTIN is required patient’s survival (Supplementary Table S4). These data suggested for NSCLC cell growth and tumorigenicity. that DSNT upregulation is one important mechanism underlying To further investigate the mechanism by which DSTN promoted human lung adenocarcinoma pathogenesis. lung adenocarcinoma cell growth, we examined the effect of DSTN on cell-cycle progression and the induction of apoptosis by flow cyto- DSTN expression promotes the proliferation of NSCLC cells metry. The results revealed that accumulated A549 cells in the G2–M in vitro and tumorigenesis in vivo via accelerating cell-cycle phase increased after knocking down DSTN expression with shDSTN, progression and inhibiting apoptosis and the similar results were also observed in H1299-shDSTN cells. fi – We rst examined the expression levels of DSTN in immortalized Inversely, ectopic DSTN expression in A549 cells decreased the G0 G1 lung epithelial cell BEAS-2B and five NSCLC cell lines, A549, H1299, phase cell population, with a corresponding increase in the S-phase H460, H1373, and H1573 by Western blot analysis (Fig. 3A). Given population. Overexpression of DSTN in knockdown cells efficiently – expression level of DSTN protein is higher in several NSCLC cell lines, decreased G2 M phase arrest (Supplementary Fig. S6A). The thus we selected to knockdown DSTN expression using short hairpin molecular mechanisms were further investigated by Western blot- RNA (shRNA) targeting DSTN in two NSCLC cell lines, A549 and ting (Supplementary Fig. S6B). The results showed that DSTN – H1299, and measure proliferation and apoptosis change in these cells knockdown arrested the cell cycle at the G2 Mphasewithan in vitro. Vector cloned with scrambled shRNA was used as control increase in cyclin B1, Wee1 protein levels, and the increased (shNC). The knockdown of DSTN was confirmed by Western blot phosphorylation of cdc2 (Thr15). In addition, Annexin V assay analysis (Fig. 3B). To rule out the role of cofilin-1, we examined the detected that DSTN knockdown in A549 and H1299 cells signif- expression level of cofilin-1 in A549- and H1299-shDSTN and control icantly increased apoptosis induction, whereas ectopic expression of cells at the same time. The results showed that DSTN downregulation DSTN inhibited both early and late apoptosis in A549 cells (Sup- did not lead to a significant change of cofilin-1 protein expression level plementary Fig. S7). The rescue experiment also showed the above (Fig. 3C). consistent results, suggesting that the oncogenic effects of DSTN We observed that the DSTN knockdown resulted in a substantially could be through cell apoptosis suppression and promotion of the reduced growth rate (Fig. 3D) and lower plating efficiency (Fig. 3E). cell-cycle progression in lung adenocarcinoma cells. Conversely, ectopic expression of DSTN in A549 and H1299 cells significantly promoted cell proliferation compared with the control DSTN expression enhances NSCLC cells metastasis and invasion cells. To further confirm the role of DSTN in NSCLC and rule out a in vitro and in vivo putative shRNA-mediated off-target effect, the rescue experiment was Tumor metastasis is the main reason for the low survival rate of lung performed by lentiviral overexpression of DSTIN in A549 and H1299 adenocarcinoma. The high expression of oncogenes is critical for cells with DSTN knockdown by sh1DSTN, which target 30UTR of tumor progression because it promotes the migration and invasiveness DSTN. The results showed that the lentiviral DSTN-expressing vector of tumor cells. The wound-healing assay showed that DSTN down- in DSTN knockdown cells efficiently rescued the proliferation ability regulation in A549 and H1299 cells caused a significantly decreased in of A549 and H1299 cells. We also examine the effects of DSTN in healing ability than the control cells in wound formation, whereas normal human bronchial epithelial cells, BEAS-2B (25). The results ectopic expression of DSTN markedly increased healing ability of showed that DSTN silencing or overexpression did not significantly A549 cells (Supplementary Fig. S8). Next, we examined the role of affect the proliferation and migration of BEAS-2B cells (Supplemen- DSTN in NSCLC cell invasion using a Matrigel invasion assay. As tary Fig. S4). It is suggested that DSTN alone seems to be insufficient shown in Fig. 4A, knockdown of DSTN markedly inhibited invasion of for the oncogenic transformation of BEAS-2B cells. In addition, to cells from migrating to the bottom chambers through Matrigel com- further assess the role of DSTN in tumorigenesis in vivo, we performed pared with the scrambled control cells. In contrast, ectopic expression subcutaneous xenograft assay in nude mice using A549 and H1299 of DSTN markedly increased invasion ability of cells by 24.1%. Ectopic cells, stably silencing DSTN by shRNA, and A549 cells with ectopic expression of DSTN eliminated the inhibitory effects of DSTN knock- expression of DSTN and control cells. DSTN knockdown significantly down on migration and invasion in A549 and H1299 cells. Statistical inhibited tumor growth. As shown in Fig. 3F, A549 cells with DSTN analysis confirmed the results. Taken together, these data demon- silencing could not form a tumor in any of the 5 mice. Tumor sizes in strated that DSTN is involved in NSCLC cell migration and invasion. the H1299-shDSTN group were smaller and weighed significantly less Given that anchorage-independent growth is one of the traits of a than controls. On the contrary, the ectopic expression of DSTN in cancer cell, we performed soft agar assays to see if inhibition or

(Continued.) C, Representative Western blotting analysis of DSTN protein expression levels in lung adenocarcinoma tissue samples (T) with lymph node metastasis or no metastasis and matched adjacent nontumor tissues (N). All tissue samples used in this experiment have the same or similar clinicopathologic characteristics except for the status of lymph node metastasis. The right panel is the quantified results by grayscale scanning. n ¼ 16; , P < 0.05. D, Representative Western blotting analysis of DSTN protein expression levels in lung adenocarcinoma tissue samples (T) with different clinical TNM stages and matched adjacent tissues (N). All tissue samples used in this experiment have the same or similar clinicopathologic characteristics except for clinical TNM stages. Right, the quantified results by grayscale scanning. n ¼ 9; P < 0.05. E, Representative positive and negative IHC staining of DSTN in lung adenocarcinoma tissues (I, II) and their adjacent nontumor tissues (III, IV; magnification, 200). F and G, Kaplan–Meier analysis of overall survival of patients with lung adenocarcinoma with different expressions of DSTN. Low (Lo) and high (Hi) expression of DSTN (F) and different clinical stage (G) was plotted against the time of overall survival.

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Figure 3. A B C The effect of DSTN on the prolifer- A549 H1299 A549 H1299 ation of LUAD cells in vitro and shNC sh1DSTN sh2DSTN sh3DSTN DSTN in vivo. A, DSTN expression level in DSTN A549 1.00 0.10 0.08 0.06 0.86 1.00 0.22 0.22 0.26 1.13 1.00 2.10 1.00 2.32 GAPDH BEAS-2B and five human NSCLC cell BEAS-2BA549 H1299 H1373 H460 H1573 Cofilin-1 lines was analyzed by Western blot DSTN DSTN 1.00 1.02 0.97 1.07 1.11 1.00 0.96 1.02 1.07 1.12 1.00 1.22 1.00 0.97 analysis. B, DSTN silencing efficiency H1299 GAPDH GAPDH of sh1DSTN, sh2DSTN, and sh3DSTN GAPDH shNC shNC Vector Vector sh1DSTN Vector sh1DSTN Vector in A549 and H1299 cell lines was sh3DSTN sh1DSTN+ sh1DSTN+ sh3DSTN sh1DSTN+ sh1DSTN+ DSTN-OE DSTN-OE DSTN-OE DSTN-OE assessed, respectively, by Western D blot analysis. C, Downregulated or 0.9 A549/shNC 0.9 H1299/shNC 1.0 A549/Vector upregulated DSTN did not affect the 0.8 0.8 expression of a cofilin-1 protein in 0.7 A549/sh3DSNT 0.7 H1299/sh3DSNT 0.8 A549/DSTN-OE 0.6 0.6 A549 and H1299 cells. D, The effect 0.6 H1299/Vector A549/sh1DSNT 0.5 H1299/sh1DSNT of DSTN knockdown and overex- 0.5 0.4 0.4 0.4 A549/sh1DSNT H1299/sh1DSNT H1299/DSTN-OE pression on cell growth was evalu- 0.3 0.3 +Vector +Vector ated by MTT assay in A549 and 0.2 0.2 0.2 OD value A549/sh1DSNT 0.1 0.1 H1299/sh1DSNT H1299 cells, respectively. E, The +DSTN-OE +DSTN-OE 0.0 0.0 0.0 effect of DSTN knockdown or over- 01 234 01 23 4 01234 expression on colony formation in Time (days) A549 and H1299 cells. The data were present as mean SD values from E sh1DSNT sh1DSNT three independent experiments. shNC sh3DSTN sh1DSNT +Vector+DSTN-OE Vector DSTN-OE Asterisks () indicate statistical sig- nificance: , P < 0.01; , P < 0.001. F, In vivo tumor formation assay of A549 DSTN knockdown or overexpression in A549 and H1299 cells on nude mice. Ectopic expression of DSTN promoted tumor growth and increased tumor volume and weight in nude mice, whereas tumor growth H1299 was inhibited by DSTN knockdown (n ¼ 5; , P < 0.05; , P < 0.001). A549 H1299 A549 H1299 400 400 600

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shNC shNC Vector Vector sh3DSTN sh1DSNT Vector sh3DSTN sh1DSNT Vector sh1DSTN+ sh1DSTN+ sh1DSTN+ sh1DSTN+ DSTN-OE DSTN-OE DSTN-OE DSTN-OE

F 600.0 H1299/shNC H1299/sh3DSTN ) 500.0 3 1,200 A549/shNC 400.0 1,000 800 300.0 A549/sh3DSTN 600 200.0 400 200 100.0 Tumor weight (mg) weight Tumor H1299/shNC 0 0.0 Tumor volume (mm volume Tumor 714212835 42 H1299/shNC H1299/sh3DSTN Time (days) H1299/sh3DSTN 900.0 A549/Vector A549/DSTN-OE 800.0 )

3 2,000 700.0 600.0 1,500 A549/Vector 500.0 400.0 1,000 300.0 A549/DSTN-OE 500 200.0 Tumor weight (mg) weight Tumor 100.0 0 0.0 Tumor volume (mm volume Tumor 714212835 42 A549/Vector A549/DSTN-OE Time (days)

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sh1DSNT sh1DSNT A shNC sh3DSTN sh1DSTN + Vector+ DSTN-OE Vector DSTN-OE A549 H1299

2,500 shNC 2,000

2,000 sh3DSTN 1,500

1, 500 sh1DSTN 1,000 1 ,000 sh1DSNT + Vector

sh1DSTN + DSTN-OE 500 500 Invading cells/ﬁeld Invading cells/ﬁeld Invading

0 0 A549 H1299 A549/Vector A549/DSTN-OE

B H1299/shNC H1299/sh3DSTN 8

6 H1299/shNC 4

H1299/sh3DSTN 2

0 1,000 μM 1,000 μM

A549/Vector nodules/mouse Metastatic shNC sh3DSTN A549/Vector A549/DSTN-OE A549/DSTN-OE 30 25 20 15 10 5

1,000 μM 1,000 μM 0 Metastatic nodules/mouse Metastatic Vector DSTN-OE

Figure 4. The effect of DSTN knockdown or overexpression on migration and invasion of A549 and H1299 in vitro and in vivo. A, The effect of DSTN on cell invasion was evaluated in A549 and H1299 cells by Matrigel invasion assay. The knockdown of DSTN in A549 and H1299 obviously decreased penetration rate through the Matrigel- coated membrane compared with control cells (scale bar, 200 mm). Statistical data represent the mean SD, and are representative of three independent experiments. , P < 0.01; , P < 0.001. B, DSTN expression promotes NSCLC cell lung metastasis in vivo. Appearance and representative images of H&E-stained sections of the lung from nude mice injected intravenously with H1299/sh3DSTN or H1299/shNC cells and A549/DSTN-OE or A549/vector cells. The arrows indicate clusters of tumor cells that have colonized in the lung (n ¼ 5, , P < 0.01; , P < 0.001).

overexpression of DSTN affects this phenotype, results showed that empty vector. Each group was assigned ﬁve mice, and at 3-weeks A549- and H1299-shDSTN cells formed less and smaller colonies, postinjection, the metastatic tumor lesion in each mouse lung was whereas the DSTN overexpression cells formed large colonies in assessed by hematoxylin and eosin staining (H&E staining). The 10 days compared with control cells (Supplementary Fig. S9). Finally, results revealed that mice with A549/DSTN-overexpression developed to determine whether DSTIN possessed oncogenic activities in vivo,a more pulmonary metastasis nodules than with the mock control. The lung metastasis model was generated by tail vein injection of nude mice nodule numbers of H1299/shNC, A549/Vector, and A549/DSTN- with NSCLC cells, H1299 cells transfected with sh3DSTN or shNC overexpressing groups were 5 1.30, 7 1.58, and 22 3.89, control vector, and A549 cells with ectopic expression of DSTN and respectively, but none in H1299/shDSTN groups (Fig. 4B). Therefore,

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our data strongly support the oncogenic nature of DSTN expression in c-Myc, cyclin D1, slug and snai1 by Western blot analysis to evaluate promoting cancer metastasis. the effect of DSTN knockdown/overexpression on the activity of Wnt/b-catenin signaling pathways. The results showed that silenc- DSTN is essential for EMT and its silencing leads to MET in NSCLC ingDSTNmarkedlydownregulatedtheexpressionofc-Myc,cyclin cells D1, slug, and snai1 in A549 and H1299 cells compared with the EMT is a critical process related to the invasion and metastasis of scramble control. Reverse results were observed in A549 cells with tumor cells. Given the frequent overexpression of DSNT in patients ectopic overexpression of DSTN (Fig. 6B), and the optical density with lung adenocarcinoma, this promoted us to investigate the role of of each protein was quantified by GAPDH optical density. More- DSTN in the regulation of alveolar EMT, the loss-of-function studied over, the above results were further confirmed by real-time PCR was conducted using shDSTN or shNC lentivirus to stably transfect using GAPDH as an internal control (Fig. 6C–E). These results A549 and H1299 cells, followed by exploring the shift of cell mor- suggest that the decrease in c-Myc, cyclin D1, slug, and snai1 phology, adhesion, and evaluating the expression of EMT markers. occurred after inhibition of the Wnt/b-catenin pathway when The two NSCLC cell lines presented a spindle-shaped, cube-, or blocking DSTN in NSCLC cells. Our results indicate that over- polygon-shaped feature, while treated with shDSTN, cancer cells were expressed DSTN could be a critical stimulating factor for the changed into flattened morphology with clearly defined intercellular activation of the Wnt/b-catenin pathway in NSCLC cells. boundaries, much more extensive volume and spreading area (Fig. 5A), accompanied by a marked reduction in the protein and DSTN overexpression promotes b-catenin nuclear translocation mRNA levels of Vimentin and N-cadherin, and marked increase of E- and enhances its interaction with TCF4 cadherin and g-catenin, as compared with the control cells (Fig. 5B; In most cases, the elevated expression of b-catenin is the cause of Supplementary Fig. S10A–S10C). In line with these observations is activating the canonical pathway. In the canonical pathway, b-catenin DSTN overexpression in A549 cells leading to a switch from epithelial- combined with LEF/TCF4 complex in the nucleus to activate like sheet to a scattered fibroblast-like appearance, accompanied Wnt signaling (32, 33). Accordingly, to gain insight into the underlying notable increase in Vimentin and N-cadherin, and marked reduction mechanisms through which DSTN knockdown inhibits Wnt/b-cate- of E-cadherin and g-catenin at the protein and mRNA levels. In nin signaling, we first examined expression and nucleus subcellular addition, evidence has shown that inhibition of cell-cell and/or cell- localization of b-catenin in DSTN knockdown and control cells using matrix adhesive function correlated with tumor cell migration, inva- immunofluorescent staining and Western blot analysis. Our results sion, and EMT (26, 27). Our current studies also observed that revealed that DSTN knockdown did not cause the significant changes knocking down DSTN in A549 and H1299 notably increase the cell’s of b-catenin expression level in A549 and H1299 cells compared with adhesive ability by manifesting an increased cell-to-cell contact and shNC control cells but led to abnormal distribution of b-catenin in cell-matrix adhesion compared with the corresponding shNC groups, cells. As shown in Supplementary Fig. S12, localization of b-catenin and ectopic expression of DSTN in A549 markedly decreased cell- was mainly in the cytoplasmic membrane of A549 and H1299 cells matrix adhesion (Fig. 5C). when blocking DSTN by Immunofluorescent staining. Similarly, Vinculin is an actin-binding protein that localizes to focal Western blot analysis further showed a decrease of b-catenin proteins adhesion and regulates cell adhesion and migration (28, 29). To in nuclear fractions of A549 and H1299 cells, which stably infected investigate the role of DSTN in focal adhesion in NSCLC cells, with shDSTN (Fig. 7A). Consistently, ectopic overexpression of DSTN immunostaining was performed using anti-Vinculin and a second- did not affect the protein level of b-catenin expression as well but ary antibody conjugated with Alexa Fluor 488. Alexa 568-Phalloidin caused b-catenin to mainly localize in the nucleus in A549 cells with was used to visualize F-actin. Nuclei were visualized using DAPI DSTN overexpression compared with vector control. Furthermore, the stain. We observed that Vinculin patches in A549 and H1299 cells lentiviral DSTN-expressing vector in DSTN knockdown cells effi- withDSTNsilencingappearlargerstreaks or dot-like structures at ciently restored nucleus subcellular localization of b-catenin. These the cell periphery than the scramble control cells. Moreover, the data thus far promoted us to ask whether DSTN interacts with number of Vinculin patches per cell was significantly increased in b-catenin in NSCLC cells. To test this possibility, we conducted A549- and H1299-shDSTN cells compared with the control reciprocal immunoprecipitation experiments. We observed that cells (Supplementary Fig. S11). These data suggest that overexpres- DSTN and b-catenin interact with each other in HEK293T cells sion DSTN could promote lung adenocarcinoma progression by coexpressing b-catenin and DSTN (Supplementary Fig. S13), and inducing EMT. interaction of endogenous b-catenin and DSTN in A549 and H1299 cells (Fig. 7B), suggesting that DSTN could be an important DSTN knockdown inhibits activity of Wnt/b-catenin pathway factor to enhance b-catenin nuclear localization. In addition, we Wnt/b-catenin signaling pathway is broadly involved in the process wonder whether DSTN expression affects the interaction of b-catenin of promoting tumorigenicity, cell stemness, cell invasiveness, and EMT with TCF4. We thus further examined the interaction of b-catenin and induction (30, 31). To gain insights into the underlying mechanism of TCF4 in A549 and H1299 cells stably infected with shDSTN or up-regulated DSTN-mediated tumorigenicity and EMT, we assessed scramble control. Immunoprecipitation results showed that knocking the activity of the Wnt/b-catenin pathway and the expression of its down DSTN expression in A549 and H1299 cells markedly impaired downstream genes. Luciferase experiment with Top-Luc Flash report- interaction of b-catenin and TCF4, whereas the interaction of b-cate- er and pRL-TK (as an internal control) showed that stable transfection nin and TCF4 was increased in A549 cells with ectopic expression of of shDSTN into A549 and H1299 cells, but not the scramble vector DSTN (Fig. 7C). Rescue experiment showed that the DSTN- shNC, significantly inhibited the activity of the TOPFlash reporter, expressing vector efficiently restored the interaction of b-catenin and without affecting the control FOPFlash reporter, whereas ectopic TCF4 in A549 and H1299 with DSTN knockdown. Taken together, expression of DSTN markedly increased the activity of the TOPFlash this study additionally identified the regulatory role of DSTN as an reporter (Fig. 6A). Next, we further investigated expression levels of activator of the Wnt/b-catenin signaling pathway, and demonstrated

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A sh1DSTN+ sh1DSTN+ shNC sh3DSTN sh1DSTN Vector DSTN-OE Vector DSTN-OE A549 H1299

B A549 H1299 A549 E-Cadherin

1.00 37.98 15.75 20.71 6.48 1.00 6.14 5.75 4.65 1.02 1.00 0.62 N-Cadherin

1.00 0.18 0.12 0.14 1.01 1.00 0.14 0.21 0.18 1.16 1.00 2.42 Vimentin

1.00 0.32 0.14 0.19 1.25 1.00 0.12 0.23 0.15 1.29 1.00 2.28 γ-catenin

1.00 11.01 4.63 5.21 1.03 1.00 2.86 2.87 2.95 1.23 1.00 0.52 GAPDH

shNC shNC Vector Vector Vector sh3DSTNsh1DSTN sh3DSTNsh1DSTN sh1DSTN+ sh1DSTN+ sh1DSTN+ sh1DSTN+ DSTN-OE DSTN-OE DSTN-OE C sh1DSTN+ sh1DSTN+ shNC sh3DSTN sh1DSTN Vector DSTN-OE Vector DSTN-OE A549 H1299

1,200 shNC 600 1,000 sh3DSTN 500 800 sh1DSTN 400 sh1DSTN + Vector 600 300 sh1DSTN + DSTN-OE 400 200 200 100 Adhesive cells/ﬁeld Adhesive 0 cells/ﬁeld Adhesive 0 A549 H1299 A549/Vector A549/DSTN-OE

Figure 5. Knocking down DSTN inhibits EMT in A549 and H1299 cells. A, DSTN knockdown induces epithelial morphology changes in A549 and H1299 cells: oval epithelial-like type with much larger volume and increase of cell–cell contact (scale bar, 100 mm). B, The effect of DSTN knockdown or overexpression on EMT marker expression, including E-cadherin, N-cadherin, Vimentin, and g-catenin, was assessed by Western blot analysis. GAPDH served as the loading control. C, Cell adhesion assay analysis of the effects of DSTN knockdown or overexpression on the adhesive ability of the A549 and H1299 cells (scale bar, 200 mm). Statistical data shown are the mean SD of three independent assays. Asterisks () indicate statistical signiﬁcance (, P < 0.001).

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A Topﬂash reporter Topﬂash reporter 1.2 2.5 shNC 1.0 2.0 sh3DSTN

0.8 sh1DSTN 1.5 sh1DSNT + Vector 0.6 1.0 sh1DSTN + DSTN-OE 0.4 0.5 0.2

Relative luciferase activity luciferase Relative 0.0 Relative luciferase activity luciferase Relative 0.0 Control A549/DSTN-OE A549 H1299 B A549H1299 A549 E

Cyclin D1 Vector A549/DSTN-OE 1.00 0.16 0.07 0.10 0.74 1.00 0.11 0.18 0.20 0.79 1.00 1.61 3.0 c-Myc 2.5 1.00 0.73 0.69 0.52 1.11 1.00 0.13 0.20 0.32 1.03 1.00 2.27 2.0 Slug 1.5 1.00 0.26 0.34 0.44 0. 92 1.00 0.71 0.24 0.66 1.43 1.00 2. 87 1.0 Snai1

1.00 0.29 0.05 0.03 0. 57 1.00 0.18 0.28 0.25 1. 19 1.00 2. 45 0.5 0.0 GAPDH

Relative mRNA expression Relative Cyclin D1 c-Myc Slug Snai1

shNC shNC Vector Vector Vector sh3DSTNsh1DSTN sh3DSTN sh1DSTN sh1DSTN+ sh1DSTN+ sh1DSTN+ sh1DSTN+ DSTN-OE DSTN-OE DSTN-OE C D

1.2 A549 1.2 H1299 1.0 shNC 1.0 shNC

0.8 sh3DSTN 0.8 sh3DSTN

0.6 sh1DSTN 0.6 sh1DSTN

0.4 sh1DSTN + Vector 0.4 sh1DSTN + Vector

0.2 sh1DSTN + DSTN-OE 0.2 sh1DSTN + DSTN-OE

0.0 0.0 Relative mRNA expression Relative Cyclin D1 c-Myc Slug Snai1 mRNA expression Relative Cyclin D1 c-Myc Slug Snai1

Figure 6. DSTN knockdown or overexpression affects Wnt target gene expression by regulating activity of the Wnt/b-catenin signaling pathway. A, The effect of DSTN knockdown or overexpression in A549 and H1299 cells on the Wnt/b-catenin signaling pathway was analyzed by dual-luciferase assays. B, Western blot analysis of the indicated protein expression in A549 and H1299 cell after stable transfection with shDSTN, DSTN-OE or control (shNC/Vector). C–E, The Effect ofDSTNknockdowninA549andH1299cellsandoverexpressioninA549onmRNA expression of c-Myc, Cyclin D1, slug, and snai1 was examined by real-time RT-PCR analysis. Statistical data are shown in A and C–E as mean SD of three independent assays (, P < 0.01; , P < 0.001). that alteration in DSTN expression led to an interchange of localization to its aberrant expression level in lung adenocarcinoma, our results pattern of b-catenin between the nucleus and cytoplasmic membrane suggest that DSTN can serve as an initiation factor and/or a prognostic in A549 and H1299 cells. biomarker of lung adenocarcinoma metastasis. Previous studies have demonstrated that actin-binding proteins (ABP), which are responsible for polymerization and treadmilling of Discussion actin, regulate actin filament dynamics (34–36). One of the most In this study, we provided strong evidence supporting that enforced important ABP families is the actin-depolymerizing factor (ADF)/ expression of DSTN is a crucial aggravating factor of lung adenocar- cofilin family, which includes destrin (DSTN), cofilin-1 and cofilin- cinoma progression and metastasis. DSTN expression is vital in 2 (37, 38). These proteins are the key regulators in regulating actin facilitating lung adenocarcinoma cell proliferation and invasion cytoskeleton remodeling, a crucial process for cytokinesis, as well as through its coordinated augment of b-catenin nuclear trans-localiza- regulating in polarization involved in cell growth, migration and tion, leading to its interaction with TCF4. Overexpression of DSTN invasion of cancer cells, but their property of promoting proliferation correlates with EMT, lymph node metastasis, late-stage lung adeno- and metastasis has not yet been reported in human lung adenocarci- carcinoma and poor survival of lung adenocarcinoma. In addition, due noma. Especially, DSTN is far less characterized in this family than

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AB Total Nucleus Cytoplasm Total Nucleus IP β-catenin

DSTN

Nuclear input IgG β -catenin Destrin IB

A549 Lamin B1 β-catenin

GAPDH DSTN

β A549 -catenin Vector Vector DSTN-OE DSTN-OE IgG DSTN β-catenin H1299 Lamin B1 DSTN GAPDH H1299

shNC shNC shNC Vector Vector Vector sh3DSTN sh1DSTN sh1DSTN sh1DSTN sh1DSTN+ sh1DSTN+ sh3DSTN sh1DSTN+ sh1DSTN+ sh3DSTN sh1DSTN+ sh1DSTN+ IgG DSTN-OE DSTN-OE DSTN-OE

C A549 H1299 A549 IP: IgG TCF4 IgG β-catenin IgG TCF4 IgG β-catenin IP: IgG TCF4 IgG β-catenin sh3DSTN −−+−−+ −−+−−+ IB DSTN-OE −−+−−+ IB β-catenin β-catenin IP TCF4 IP TCF4 IgG IgG β-catenin β-catenin

Input TCF4 Input TCF4 DSTN DSTN

GAPDH GAPDH

A549 H1299 IgG TCF4 IgG β-catenin IgG TCF4 IgG β-catenin

IB shNC sh1DSTN sh1DSTN+ Vector sh1DSTN+ DSTN-OE shNC sh1DSTN sh1DSTN+ Vector sh1DSTN+ DSTN-OE shNC sh1DSTN sh1DSTN+ Vector sh1DSTN+ DSTN-OE shNC sh1DSTN sh1DSTN+ Vector sh1DSTN+ DSTN-OE β-catenin

IP TCF4

IgG β-catenin

TCF4 Input DSTN

GAPDH

Figure 7. DSTN knockdown or overexpression affects the subcellular localization of b-catenin and its interaction with TCF4 in A549 and H1299 cells. A, The effect of DSTN knockdown or overexpression on expression and cellular distribution of b-catenin in A549 and H1299 cells was assessed by Western blot analysis. B, Co-IP showed an interaction between endogenous b-catenin and DSTN in A549 and H1299 cells. Cell lysate was incubated with an anti-b-catenin or anti-DSTN antibody for the IP experiment. IgG was used as a negative control. C, The IP experiment using anti-b-catenin or anti-TCF4 antibody showed that DSTN knockdown inhibited the interaction of endogenous b-catenin and TCF4 in A549 and H1299 cells, whereas DSTN overexpression increased the interaction of endogenous b-catenin and TCF4 in A549 cells.

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other numbers; Moreover, it remained unknown whether DSTN which locates in cell–cell adherent junctions at the membrane. In involved in EMT and/or how it aggravates lung adenocarcinoma tumor cells, activated b-catenin can translocate to the nucleus and progression. interacts with TCF/LEF transcription factors to activate transcription We found a dramatic elevation of DSTN in tumor tissues of the of Wnt targeting genes. In addition, it has been reported that EMT also mouse lung cancer model compared with control mice using iTRAQ induces a switch from the b-catenin/E-cadherin complex to the analysis. Consistent with this, a frequently upregulation of DSTN b-catenin/TCF4/Twist1 complex, which then binds to cancer stem expression in human lung adenocarcinoma tissues was identified. cell–related gene promoters to activate oncogene expression (46). Our Simultaneously, we observed that DSTN upregulation was associated studies show that silencing DSTN promotes apoptosis and induces G2– with the progression and liver metastasis of lung cancer in the mouse M arrest in NSCLC cells. Moreover, DSTN knockdown could induce model. Moreover, high DSTN expression in tumors was significantly the abnormal intracellular distribution of b-catenin and the expression correlated with lymph node metastasis of lung adenocarcinoma, dysregulation of multiple downstream genes of Wnt/b-catenin, such as clinical TMN stage, and worse overall survival of patients with clinical c-myc, cyclin D1, slug, and snai1 in A549 and H1299 cells. Immu- lung adenocarcinoma. In lines with our findings, upregulated DSTN nofluorescence assay showed that b-catenin is mainly anchored on the has been found in pancreatic cancer as well (16). Conversely, DSTN cytoplasmic membrane and its nuclear distribution was significantly knockdown markedly inhibits migration and invasion of human colon decreased in DSTN-knockdown cells (Fig. 7A). More interestingly, a cancer Isreco1 cells (15). These data add support to our animal model co-IP assay of the nuclear fraction revealed that DSTN is associated data, clinical, and cellular model data, suggesting that DSTN may play with b-catenin in the nucleus. We found that this interaction increased a critical role in the initiation and/or progression of lung adenocar- the translocation ability of b-catenin to the nucleus in A549 cells with cinoma. Moreover, it may serve as a potential therapeutic/gene therapy the enforced expression of DSTN. Moreover, further analysis con- target, as well as a biomarker to predict the progression and prognosis firmed that DSTN depletion affects the interaction of b-catenin with of lung adenocarcinoma in the future. TCF4, leading to a decrease in the complex formation of b-catenin– DSTN (also known as an actin-depolymerization factor, ADF) is an TCF4 transcription factor in NSCLC cells. These results promote us to essential actin regulatory protein of the ADF/cofilin family. Mechan- postulate that DSTN overexpression contributes to nuclear entry of ically, it binds to the subunits of filamentous actin, enhances the b-catenin and the formation of b-catenin–TCF4 transcription factor subunit off-rate and promotes filaments severing, which is required for complex; and the latter is an important process in the development of tumor cell migration and invasion (11, 12). However, the mechanism NSCLC metastatic potential and EMT phenotype (31, 47). Therefore, by which DSTN contributes to the tumor has been still poorly our findings provide new insights into the roles of DSTN that facilitate understood. Experimental and clinical studies have shown that the invasion and metastasis of lung adenocarcinoma, suggesting DSTN EMT process plays a gatekeeper role in tumor cells invasion, metastasis may promote NSCLC cell proliferation and metastasis by activating and even chemoresistance (39, 40). Overexpression DSTN enhances Wnt/b-catenin signaling pathway. NSCLC cell migration and invasion probably through the EMT, In summary, we not only provide evidence that DSTN participated because DSTN knockdown in NSCLC cells results in lowered levels in lung adenocarcinoma development and progression, and its over- of Vimentin and N-cadherin but an increase of E-cadherin and expression indicated a poor survival of patients with lung adenocar- g-catenin levels. However, overexpression of DSNT in A549 reduced cinoma, but also illustrated the critical role of the interaction between E-cadherin and g-catenin expression and increased levels of Vimentin DSTN and b-catenin, by which EMT is established in cancer metas- and N-cadherin expression, indicating a positive correlation between tasis. Collectively, we showed the importance of DSTN in lung DSTN and EMT, which might account for the potential invasion and adenocarcinoma malignancy and proposed that DSTN is a potential metastasis of those cells with a high level of DSTN. In addition, our therapeutic target and a biomarker for lung adenocarcinoma. The results also showed that overexpressed DSTN in A549 cells increased proposed DSTN–b-catenin axis may offer a new avenue to develop slug, snai1 mRNA and protein levels (important transcriptional novel approaches anti-lung adenocarcinoma in the future. repressor of E-cadherin and inducer of EMT), while DSTN knockdown markedly decreased slug and snai1 expression levels. It has been Disclosure of Potential Conflicts of Interest well-known that E-cadherin and Vimentin are the major regulators No potential conflicts of interest were disclosed. involved in the EMT process. Moreover, E-cadherin downregulation and/or Vimentin upregulation are cornerstones for the initiation of Authors’ Contributions EMT, the gain of invasion and installation of metastasis of cancer H.-J. Zhang: Investigation. W.-J. Chang: Investigation. C.-Y. Jia: Methodology. cells (41, 42). We speculated that DSTN-dependent expression of EMT L. Qiao: Data curation. J. Zhou: Investigation. Q. Chen: Investigation. X.-W. Zheng: Resources. J.-H. Zhang: Resources. H.-C. Li: Resources. Z.-Y. Yang: Methodology. relevant proteins might be a potential mechanism, by which they lead Z.-H. Liu: Software. G.-C. Liu: Resources. S.-P. Ji: Formal analysis, funding to lung adenocarcinoma metastasis. acquisition, writing-review and editing. F. Lu: Conceptualization, formal analysis, It is well established that a variety of signaling pathways is involved supervision, funding acquisition, writing-original draft, project administration, in the activation of EMT programs, including Wnt/b-catenin signal- writing-review and editing. ing (43). However, the physical and biological relationships between EMT and the Wnt/b-catenin pathway have not been thoroughly Acknowledgments understood. Wnt/b-catenin signaling is tightly regulated at multiple This work was supported by National Natural Science Foundation of China (no. cellular levels and is dysregulated in numerous types of cancer, 81372147) and Henan University support grant CX3070A0780502. including lung cancer (44). Abnormal activation of the Wnt/b-catenin The costs of publication of this article were defrayed in part by the payment of page pathway is a crucial oncogenic step to tumor initiation and progres- charges. This article must therefore be hereby marked advertisement in accordance sion (45). b-catenin is a key component of Wnt/b-catenin signaling. with 18 U.S.C. Section 1734 solely to indicate this fact. Moreover, it is a dual-function protein that is involved in transcriptional regulation and cell–cell adhesion. In normal epithelial cells, Received February 25, 2020; revised July 9, 2020; accepted August 25, 2020; b-catenin interacts with E-cadherin to form an adhesion complex, published first September 2, 2020.

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OF14 Mol Cancer Res; 2020 MOLECULAR CANCER RESEARCH

Destrin Contributes to Lung Adenocarcinoma Progression by Activating Wnt/ β-Catenin Signaling Pathway

Hui-Juan Zhang, Wen-Jing Chang, Cai-Yun Jia, et al.

Mol Cancer Res Published OnlineFirst September 2, 2020.

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