Destrin Contributes to Lung Adenocarcinoma Progression By

Author Manuscript Published OnlineFirst on September 2, 2020; DOI: 10.1158/1541-7786.MCR-20-0187 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Destrin contributes to lung adenocarcinoma progression by

activating Wnt/β-catenin signaling pathway

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*& Feng Lu1*

1Joint National Laboratory for Antibody Drug Engineering, Henan University, Kaifeng 475004, PR China;

2Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng 475004, PR

China;

3Laboratory for NanoMedical Photonics, School of Basic Medical Science, Henan University, Kaifeng 475004, PR

China;

4Department of clinical laboratory, Puyang hospital of traditional Chinese medicine, Puyang 457001,PR China;

5Department of Pathology, Puyang Oilfeld General Hospital, Puyang 457001, PR China)

# These authors have equally contributed to this work

* Correspondence is addressed to these authors at Joint national laboratory for antibody drug

engineering, Henan university, PR China

Tel: 86-371-23880398 ； Fax: 86-371-23880398; e-mail: [email protected];

[email protected]

Running title: Oncogenic role of destrin in lung adenocarcinoma

Key words: lung adenocarcinoma; Destrin; β-catenin; metastasis; EMT

Conflict of interest

The authors declare that they have no conflict of interest.

Downloaded from mcr.aacrjournals.org on September 28, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 2, 2020; DOI: 10.1158/1541-7786.MCR-20-0187 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Lung cancer, especially lung adenocarcinoma (LUAD), is one of the most common neoplasms worldwide. However, the mechanisms underlying its initiation, development, and metastasis are still poorly understood. Destrin (DSTN) is a member of ADF/cofilin family. Its detailed biological function remains unknown, although it is reported that DSTN is involved in cytoskeleton remodeling and regulation of actin filament turnover. Recent evidence has shown that high expression of cofilin-1 is associated with invasion and poor prognosis of several types of human tumors, but the detailed mechanism is still entirely unclear, particularly in lung cancer tumorigenesis and malignancy. Here, we report that DSTN was highly expressed in a mouse lung cancer model induced by urethane and in clinical LUAD tissue samples. Its expression level was positively correlated with cancer development, as well as metastasis to the liver and lymph nodes.

Consistently, it was directly associated with the poor prognosis of LUAD patients. Furthermore, we also found that DSTN promotes cell proliferation, invasion and migration in vitro, and facilitates subcutaneous tumor formation and lung metastasis via intravenous injection in vivo.

Mechanically, DSTN associates with and facilitates nuclear translocation of β-catenin, which promotes epithelial-mesenchymal transition (EMT). Taken together, our results indicated that

DSTN enhances lung cancer malignancy through facilitating β-catenin nuclear translocation and inducing EMT. Combined with multivariate analyses, DSTN might potentially serve as a therapeutic target and an independent prognostic marker of LUAD.

Implications: This finding indicated DSTN facilitates β-catenin nuclear translocation and promotes malignancy in lung adenocarcinoma.

Introduction

Lung cancer is the leading cause of cancer deaths in China and worldwide. Despite a great progress in cancer research and therapeutic strategies over the last decades, the prognosis of lung cancer remains poor with a 5-year survival rate of less than 15% (1, 2). Therefore, both understanding the mechanism of cancer development and progression and early diagnosis are critical to lung cancer treatment. Unfortunately, because of the lack of early and reliable diagnostic biomarkers together with the limited understanding of its carcinogenic mechanisms, more than 70% of the patients are found in advanced stages accompanied by extensive invasion and metastasis, which results in the loss of opportunity for curative surgical resection and a poor prognosis (3).

Lung adenocarcinoma (LUAD) is the most prevalent histological type of lung cancer, which accounts for ~40% of all lung cancers. During the last few years, LUAD has an increasing frequency, and its prognosis has not been obviously improved (4). The prognosis of LUAD is highly associated with lymph node and distant metastasis. Therefore, it is of great interest to identify novel biomarkers that serve for early diagnosis and accurate prognosis prediction, which allow improving the prognosis.

Proteomic technologies have been widely used in the global analysis for lung cancer biomarker discovery (5). Although many proteomic studies on LUAD 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 clinically established biomarkers available for early detection of LUAD. Comparative proteomics analysis of successive stages of human LUAD is the most direct and persuasive way to identify biomarkers for early diagnosis of LUAD. However, a major obstacle in the analysis of tissue specimens is tissue heterogeneity, in which different cells are collected. Furthermore, it is difficult to obtain a large amount of early-stage clinical lung adenocarcinoma tissue.

To search for the early biomarkers and explore the underlying mechanisms of initiation and metastasis of LUAD, in this study, we developed a urethane-induced mouse lung adenocarcinoma model, which exhibits similar histological appearance and molecular changes to human lung adenocarcinoma (8, 9). We used a laser capture microdissection (LCM) approach to purify the

target cells from lung tissues of a mouse model with early carcinogenesis lesion, then investigated the differential proteins in tumor and control tissue using isobaric tagging for relative and absolute protein quantification (iTRAQ) combined with two-dimensional liquid chromatography-tandem mass spectrometry/mass spectrometry (2D-LC-MS/MS) approach. Subsequently, we further validated our observations by immunohistochemistry (IHC) and Western blot analysis. Among the identified 191 potential proteins, destrin (DSTN) protein expression (Dstn, tr|Q4FK36|) displayed a significant up-regulation (3.67 folds to control) in early-stage mouse lung cancer tissues compared with the control group.

DSTN belongs to the ADF/cofilin family, consisting of DSTN, cofilin-1, and muscle-specific cofilin-2 (10). These proteins are abundant and essential in almost all eukaryotic cell types. They involve in cytoskeleton remodeling and precise regulation of the actin filament turnover, take part in numerous cellular processes such as cell cytokinesis, proliferation, and membrane trafficking

(11, 12). Recent studies showed that actin dynamics and their regulation of target proteins are not only essential for the healthy development and function of the cells, but also play a crucial role in human diseases, including cancers. However, most research on ADF/cofilin family proteins in metastatic invasion has focused on cofilin-1(13, 14). As a member of the ADF/cofilin protein family, the DSTN with the least study, and is only found to regulate the migration and invasion of colon cancer through regulating action dynamics (15). Its high expression was the association with growth and perineural invasion of pancreatic cancer (16). However, its precise roles in human cancers, particularly in LUAD, and its downstream signaling pathway in these processes, remain largely unclear.

Here, we have found that overexpressed DSTN seems to be a functional oncogenic molecule at

LUAD both in vitro and in vivo. For the first time, we have demonstrated that DSTN expression is associated with progression of lung cancer development and liver metastasis in urethane-induced lung adenocarcinoma model. Immunohistochemistry and Western blot analysis showed that the high expression level of DSTN is closely related to lymph node metastasis and aggravates the clinical progression of human lung adenocarcinoma. The knockdown of DSTN inhibits cell proliferation, migration, and invasion, leading to an arrest of the cell cycle, and in turn promotes apoptosis of NSCLC cells. Performing the rescue experiment by lentiviral overexpression of

DSTN in knockdown cells, efficiently restored cell proliferation and invasion et al. Furthermore, 4

we observed that overexpressed DSTN could up-regulate the activity of the Wnt/β-catenin signaling pathway by promoting nuclear trans-localization of β-catenin and interaction of

β-catenin with TCF4. These observations provide new insights into the molecular function of

DSTN as well as its aggravating mechanisms in the development and progression of lung adenocarcinoma.

Materials and Methods

Reagent and antibodies

Cell culture media, reagent and antibody information were presented in the Supplementary Data.

Urethane-induced murine lung adenocarcinoma model

Six-week-old female BALB/c mice (Weitong Lihua Animal Co., Beijing, China) were housed under pathogen-free conditions and all procedures involving mice followed Henan University

Animal Care and Use Committee guideline. The urethane-treated group mice received an intraperitoneal injection of urethane (800 mg/kg body weight; Sigma, St Louis, MO, USA) dissolved in sterile 0.9% NaCl saline once a week for 12 consecutive weeks. Control group mice received parallel intraperitoneal injection of saline (17). Animals were sacrificed at 16, 20, 24, 28 and 32 weeks after the first urethane injection with age-matched controls, and their lungs were prepared for histological, iTRAQ and Western blotting analysis. Five mice in each group were examined at each time point.

Sample preparation and LC-ESI-MS/MS analysis

Lung tissue protein sample preparation, iTRAQ labeling, LC-ESI-MS/MS analysis and protein identification were performed as described previously (18, 19). Additional details are provided in the Supplementary Data.

Clinical lung adenocarcinoma and adjacent no-tumor lung tissues

A total of 30 paired samples of lung adenocarcinoma and adjacent no-tumor lung tissues were obtained from patients who underwent surgery between 2013 and 2015 and preserved in the

Department of Pathology, Puyang Oilfield General Hospital，Henan, PR China. All patients gave written informed consent before sample collection. None of the lung adenocarcinoma patients received radio- or chemotherapy before surgery. Lung adenocarcinoma tissue microarray

(HLugA180Su02) purchased from Shanghai Outdo Biotech Co., Ltd. included 86 lung adenocarcinoma patients undergoing surgery between 2004 and 2009. This study was approved by the Ethic Committee of the Medical School, Henan University, China and all methods in this study were carried out in accordance with the recommended guidelines.

Cell culture, expression plasmids, and stable transfection of shRNA

The human NSCLC cell lines, A549, HCI-H1299, HCI-H460, HCI-H1373, HCI-H1573 and immortalized lung epithelial cell line BEAS-2B were obtained from the Cell Bank of Type Culture

Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were routinely screened to confirm Mycoplasma-negative status, and cell line was authenticated by examination of morphology, growth characteristics and short tandem repeat analysis. There were enough frozen vials for each cell line to ensure that all cell-based experiments were carried out in the cells under test and cultured for 8 weeks or less. The cells were routinely cultured in RPMI1640 medium

(Corning, USA) supplemented with 10% fetal bovine serum (PAN Biotech, Germany), 100U/mL penicillin and 100μg/mL streptomycin in a humidified cell incubator with an atmosphere of 5%

CO2 at 37°C (all cells are cultured in the same condition). Empty vector pcDNA3.1(+), pCDH-CMV-MCS-EF1-Neo and pCDH-CMV-MCS-EF1-puro were from YouBio (Hunan, China).

The detail information of cell culture and plasmids construction are described in the

Supplementary Materials and Methods.

RNA extraction and qRT-PCR analysis

Total RNA was extracted from frozen tissues or cell lines using Trizol reagent (Takara, Dalian,

China) according to the manufacturer’s instructions. For reverse transcriptions, 1μg of total RNA was used with Reverse Transcriptase Kit (RR036A, Takara). Real-time PCR was performed using the TB GreenTM premix Ex TaqTM (RR420A, Takara) and ABI Prism 7900 System. The expression levels of genes were calculated using the 2-ΔΔCt method, and GAPDH was used as endogenous control (20). The qRT-PCR primers used in this study were presented in Supplementary Table 6.

Rescue experiment

To recover the expression of DSTN-silenced cells, A549 or H1299 cells with DSTN knockdown by sh1DSTN targeting 3’UTR of DSTN were transduced with lentiviral DSTN-overexpression vector that was bearing the resistance to Neo. Cells then were selected for 7 days with G418, then allowed to recover for 5 days, seeded and allowed to grow for 5 days. 6

Co-immunoprecipitation assay

The total Cell protein was generated by lysing cells with RIPA buffer for 1 hour at 4℃. The nuclear extraction was prepared using a NE-PER Nuclear Cytoplasmic Extraction Reagent kit

(Pierce, Rockford, IL, USA) according to the manufacturer’s instruction. Co-immunoprecipitation

(Co-IP) assay was then performed as described previously (21, 22). Protein lysates were incubated with 5μg of indicated primary antibodies overnight on a rotator at 4ºC, followed by adding 50μL of equilibrated Protein A/G beads (Santa Cruz) and incubation for another 3 h on a rotator at 4ºC.

The agarose beads were collected by centrifuging, followed by washing with lysis buffer to remove all of the non-specifically bounded proteins. The bounded immune complexes were eluted from the beads with corresponding elution buffer (elution is not necessary), boiled with sample-loading buffer, and detected by Western blotting analysis.

Additional methods

Immunohistochemistry, immunoblotting, immunofluorescence, Luciferase reporter gene assay, in vivo tumorigenesis and metastasis assay, and cell adhesion assay were performed using standard techniques. Invasion ability of cells was estimated by Matrigel invasion, Wound-healing assay and soft agar assay. Proliferation was measured by colony formation and MTT assay. The flow-cytometry analysis was used to assess cell cycle distribution and levels of cell apoptosis.

Additional details are included in Supplementary Data.

Statistical analysis

All the statistical data are presented as the mean ± standard deviation of at least three independent experiments performed in triplicate. The significance of the difference between groups was assessed with a two-tailed t-test or chi-square analysis. Kaplan-Meyer survival analysis and the log-rank nonparametric test were used to evaluate the overall survival of LUAD patients concerning DSTN expression. The effect of clinical variables on LUAD patient survival was analyzed using Cox proportional hazards regression analysis. p＜0.05 was considered statistically significant. We did all calculations and analyses with SPSS statistical software (version 20.0,

Chicago, Ⅲ).

Results

Identification of differentially expressed proteins in mouse lung tumor model

We first generated a mouse lung tumor model induced by urethane (Suppl. Fig. 1A-F). These histologic indicators of tumor progression were consistent with studies reported by others (23, 24).

We found 191 differently expressed proteins by iTRAQ analysis (Suppl. Table 1). To verify the results of mass spectrometry, we selected several differentially expressed proteins, such as Prdx5, Ctsd,

Ctsh, Ctnnal, Jup and DSTN, etc. for immunohistochemistry and Western blot analysis (Suppl. Table 2).

Representative experimental results were presented in Supplementary figure 2 and figure 3. Moreover, we observed that destrin (Dstn, tr|Q4FK36|) expression was markedly high compared to the control group (3.67-fold) (Suppl. Fig. 1G), and its link to lung adenocarcinoma had not previously been reported. We further focus on assessing its potential as a biomarker for lung adenocarcinomas and related molecular mechanisms.

DSTN aggravates tumor progression and liver metastasis in urethane-induced a mouse model of lung tumor

Immunohistochemistry was performed using antibodies against DSTN（ab186754） to measure

DSTN expression levels. The results demonstrated that DSTN protein expression increased in a stepwise manner following the progression of lung lesion (Fig. 1A-E). Furthermore, the increasing

DSTN protein expression in the mouse model’s lung tumor tissues was also confirmed by Western blot analysis (Fig. 1F). Noticeably, at the end of the experiment (32th week post-treatment of urethane), concomitant liver metastases of tumors were observed in 60% (7/10) of the mice treated with urethane while no metastasis (0/10) was detected in the matched control group (Fig. 1G). To further evaluate whether DSTN up-regulation was associated with tumor metastasis at tumor late stage, Western blot analysis was performed. As demonstrated in Fig. 1H, DSTN protein expression level was much higher in lung tumor tissues of a mouse model with liver metastasis than mice without liver metastasis. These results suggested that DSTN was involved in the progression and metastasis of LUAD in the lung cancer model induced by urethane.

DSTN was frequently up-regulated in human lung adenocarcinoma tissues and associated with poor prognosis of LUAD

To determine the expression level of DSTN in human lung adenocarcinoma, we first examined the

DSTN expression pattern in 20 pairs of human lung adenocarcinoma tissues and their adjacent non-tumor tissues by Western blot analysis. As shown in Fig. 2A, DSTN levels were noticeable 8

higher in the tumor tissues (T) than in the matched adjacent non-tumor tissues (N) (p＜0.05).

Real-time PCR assays also observed that the mRNA levels of DSTN were highly expressed in

55.2% tumor tissues (11/20) (Fig. 2B). In addition, we also further examined the association of

DSTN protein levels with lymph node metastasis and different clinical TNM stages of LUAD using Western blotting analysis. As shown in Fig. 2C, the expression of DSTN protein levels in

LUAD patients with lymph node metastasis was markedly higher than the patient with no lymph node metastasis (p＜0.05). Moreover, with the progression of clinical stages, DSTN expression increased in a stepwise manner evidenced by Western blotting analysis (Fig. 2D). Next, to further assess the protein levels of DSTN in LUAD tissues, immunohistochemistry (IHC) staining of

DSTN was performed using the LUAD tissue microarray, which containing 86 human LUAD specimens and their adjacent non-tumor tissues with complete clinicopathological characteristics and follow-up data. As shown in Fig. 2E, DSTN was detected in the cytoplasm and nucleus of tumor cells and was strongly expressed in 55.8% (46/86) tumor tissues, whereas 88.4% (76/86) adjacent non-tumor tissues showed no or low DSTN expression and only 11.6% (10/86) samples exhibited high DSTN expression. Finally, to characterize the roles of DSTN in human LUAD development and progression, the relationships between the DSTN protein expression and clinicopathological parameters of LUAD patients were analyzed. We found that high expression of

DSTN in LUAD was significantly correlated with late clinical stages (p=0.008) and with lymph node metastasis (p=0.009). No significant correlations were observed between DSTN expression and other clinicopathological characteristics, such as gender, age, tumor size and pathological stages (Suppl. Table 3).

In addition, Kaplan-Meier analysis showed that LUAD patients with high DSTN expression exhibited poorer survival than those with low expression (Fig. 2F). Moreover, we further stratified analysis and found that LUAD patients at the clinic early stage with high DSTN survived significantly shorter than those with low DSTN expression (p=0.006) (Fig. 2G). Cox multivariate analysis showed that lymph node metastasis (HR=2.850, p=0.021) and DSTN high expression

(HR=2.149, p=0.007) were negatively correlated with postoperative survival and positively correlated with mortality. While DSTN low expression favored the patient’s survival (Suppl. Table

4). These data suggested that DSNT up-regulation is one important mechanism underlying human

LUAD pathogenesis. 9

DSTN expression promotes the proliferation of NSCLC cells in vitro and tumorigenesis in vivo via accelerating cell-cycle progression and inhibiting apoptosis.

We first examined the expression levels of DSTN in immortalized lung epithelial cell BEAS-2B and five NSCLC cell lines, A549, H1299, H460, H1373 and H1573 by Western blot (Fig. 3A).

Given expression level of DSTN protein is higher in several NSCLC cell lines, thus we selected to knock down DSTN expression using short hairpin RNA (shRNA) targeting DSTN in two non-small cell lung cancer (NSCLC) cell lines, A549 and H1299, and measure proliferation and apoptosis change in these cells in vitro. Vector cloned with scrambled shRNA was used as control

(shNC). The knockdown of DSTN was confirmed by Western blot (Fig. 3B). To rule out the role of cofilin-1, we examined the expression level of cofilin-1 in A549- and H1299-shDSTN and control cells at the same time. The results showed that DSTN downregulation did not lead to a significant change of cofilin-1 protein expression level (Fig. 3C).

We observed that the DSTN knockdown resulted in a substantially reduced growth rate (Fig. 3D) and lower plating efficiency (Fig. 3E). Conversely, ectopic expression of DSTN in A549 and

H1299 cells significantly promoted cell proliferation compared with the control cells. To further confirm the role of DSTN in NSCLC and rule out a putative shRNA-mediated off-target effect, the rescue experiment was performed by lentiviral overexpression of DSTIN in A549 and H1299 cells with DSTN knockdown by sh1DSTN, which target 3’UTR of DSTN. The results showed that the lentiviral DSTN-expressing vector in DSTN knockdown cells efficiently rescued the proliferation ability of A549 and H1299 cells. We also examine the effects of DSTN in normal human bronchial epithelial cells, BEAS-2B (25). The results showed that DSTN silencing or overexpression did not significantly affect the proliferation and migration of BEAS-2B cells (Suppl. Fig. 4). It is suggested that DSTN alone seems to be insufficient for the oncogenic transformation of BEAS-2B cells. In addition, to further assess the role of DSTN in tumorigenesis in vivo, we performed subcutaneous xenograft assay in nude mice using A549 and H1299 cells, stably silencing DSTN by shRNA, and A549 cells with ectopic expression of DSTN and control cells. DSTN knockdown significantly inhibited tumor growth. As shown in Fig. 3F, A549 cells with DSTN silencing could not form a tumor in any of the 5 mice. Tumor sizes in the H1299-shDSTN group were smaller and weighed significantly less than controls. On the contrary, the ectopic expression of DSTN in A549 10

cells significantly increased tumor growth. As evidenced by a significant increase in the average size and weight of tumors. Consistent with this observation, Ki-67 proliferation assays showed that to compare with control tumors, overexpression of DSTN tumor cells showed an increased cell proliferation rate. In contrast, cell proliferation rate was significantly inhibited by DSTN knockdown (Suppl. Fig. 5). These data together suggest that DSTIN is required for NSCLC cell growth and tumorigenicity.

To further investigate the mechanism by which DSTN promoted LUAD cell growth, we examined the effect of DSTN on cell-cycle progression and the induction of apoptosis by flow cytometry.

The results revealed that accumulated A549 cells in the G2/M phase increased after knocking down DSTN expression with shDSTN, and the similar results were also observed in

H1299-shDSTN cells. Inversely, ectopic DSTN expression in A549 cells decreased the G0 to G1 phase cell population, with a corresponding increase in the S-phase population. Overexpression of

DSTN in knockdown cells efficiently decreased G2/M phase arrest (Suppl. Fig. 6A). The molecular mechanisms were further investigated by Western blotting (Suppl. Fig. 6B). The results showed that DSTN knockdown arrested the cell cycle at the G2/M phase with an increase in cyclin B1, Wee1 protein levels and the increased phosphorylation of cdc2 (Thr15). In addition,

Annexin V assay detected that DSTN knockdown in A549 and H1299 cells significantly increased apoptosis induction, whereas ectopic expression of DSTN inhibited both early and late apoptosis in A549 cells (Suppl. Fig. 7). The rescue experiment also showed the above consistent results, suggesting that the oncogenic effects of DSTN could be through cell apoptosis suppression and promotion of the cell-cycle progression in LUAD cells.

DSTN expression enhances NSCLC cells metastasis and invasion in vitro and in vivo

Tumor metastasis is the main reason for the low survival rate of lung adenocarcinoma. The high expression of oncogenes is critical for tumor progression because it promotes the migration and invasiveness of tumor cells. The wound-healing assay showed that DSTN downregulation in A549 and H1299 cells caused a significantly decreased in healing ability than the control cells in wound formation, whereas ectopic expression of DSTN markedly increased healing ability of A549 cells

(Suppl. Fig. 8). Next, we examined the role of DSTN in NSCLC cell invasion using a Matrigel invasion assay. As shown in Fig. 4A, knockdown of DSTN markedly inhibited invasion of cells 11

from migrating to the lower chambers through Matrigel compared with the scrambled control cells.

In contrast, ectopic expression of DSTN markedly increased invasion ability of cells by 24.1%.

Ectopic expression of DSTN eliminated the inhibitory effects of DSTN knockdown on migration and invasion in A549 and H1299 cells. Statistical analysis confirmed the results. Taken together, these data demonstrated that DSTN is involved in NSCLC cell migration and invasion.

Given that anchorage-independent growth is one of the traits of a cancer cell, we performed soft agar assays to see if inhibition or overexpression of DSTN affects this phenotype, results showed that A549- and H1299-shDSTN cells formed less and smaller colonies, whereas the DSTN overexpression cells formed large colonies in 10 days compared to control cells (Suppl. Fig. 9).

Finally, to determine whether DSTIN possessed oncogenic activities in vivo, a lung-metastasis model was generated by tail vein injection of nude mice with NSCLC cells, H1299 cells transfected with sh3DSTN or shNC control vector, and A549 cells with ectopic expression of

DSTN and empty vector. Each group was assigned five mice, and at 3-weeks post-injection, the metastatic tumor lesion in each mouse lung was assessed by Haematoxylin and eosin staining (HE staining). The results revealed that mice with A549/DSTN-OE developed more pulmonary metastasis nodules than with the mock control. The nodule numbers of H1299/shNC, A549/Vector and A549/DSTN-OE groups were 5±1.30, 7±1.58 and 22±3.89, respectively, but none in

H1299/shDSTN groups (Fig. 4B). Therefore, our data strongly support the oncogenic nature of

DSTN expression in promoting cancer metastasis.

DSTN is essential for EMT and its silencing leads to MET in NSCLC cells

Epithelial to mesenchymal transition is a critical process related to the invasion and metastasis of tumor cells. Given the frequent overexpression of DSNT in LUAD patients, this promoted us to investigate the role of DSTN in the regulation of alveolar EMT, the loss-of-function studied was conducted using shDSTN or shNC lentivirus to stably transfect A549 and H1299 cells, followed by exploring the shift of cell morphology, adhesion and evaluating the expression of EMT markers.

The two NSCLC cell lines presented a spindle-shape, cube or polygon shape feature, while treated with shDSTN, cancer cells were changed into flattened morphology with clearly defined intercellular boundaries, much more extensive volume and spreading area (Fig. 5A), accompanied by a marked reduction in the protein and mRNA levels of Vimentin and N-cadherin, and marked increase of E-cadherin and γ-catenin, as compared with the control cells (Fig. 5B; Suppl. Fig. 12

10A-C). In line with these observations is DSTN overexpression in A549 cells leading to a switch from epithelial-like sheet to a scattered fibroblast-like appearance, accompanied notable increase in Vimentin and N-cadherin, and marked reduction of E-cadherin and γ-catenin at the protein and mRNA levels. In addition, evidence has shown that inhibition of cell-cell and/or cell-matrix adhesive function correlated with tumor cell migration, invasion and EMT (26, 27). Our present studies also observed that knocking down DSTN in A549 and H1299 notably increase the cell’s adhesive ability by manifesting an increased cell-to-cell contact and cell-matrix adhesion compared to the corresponding shNC groups, and ectopic expression of DSTN in A549 markedly decreased cell-matrix adhesion (Fig. 5C).

Vinculin is an actin-binding protein that localizes to focal adhesion and regulates cell adhesion and migration (28, 29). To investigate the role of DSTN in focal adhesion in NSCLC cells, immunostaining was performed using anti-Vinculin and a secondary antibody conjugated with

Alexa Fluor 488. Alexa568-Phalloidin was used to visualize F-actin. Nuclei were visualized using

DAPI stain. We observed that Vinculin patches in A549 and H1299 cells with DSTN silencing appear larger streaks or dot-like structures at the cell periphery than the scramble control cells.

Moreover, the number of Vinculin patches per cell was significantly increased in A549- and

H1299-shDSTN cells compared with the control cells (Suppl. Fig. 11). These data suggest that overexpression DSTN could promote lung adenocarcinoma progression by inducing EMT.

DSTN knockdown inhibits activity of Wnt/β-catenin pathway

Wnt/β-catenin signaling pathway is broadly involved in the process of promoting tumorigenicity, cell stemness, cell invasiveness and EMT induction (30, 31). To gain insights into the underlying mechanism of up-regulated DSTN-mediated tumorigenicity and EMT, we assessed the activity of the Wnt/β-catenin pathway and the expression of its downstream genes. Luciferase experiment with Top-Luc Flash reporter and pRL-TK (as an internal control) showed that stable transfection of shDSTN into A549 and H1299 cells, but not the scramble vector shNC, significantly inhibited the activity of the TOPFlash reporter, without affecting the control FOPFlash reporter, whereas ectopic expression of DSTN markedly increased the activity of the TOPFlash reporter (Fig. 6A).

Next, we further investigated expression levels of c-Myc, cyclin D1, slug and snai1 by Western blot to evaluate the effect of DSTN knockdown /overexpression on the activity of Wnt/β-catenin signaling pathways. The results showed that silencing DSTN markedly downregulated the 13

expression of c-Myc, cyclin D1, slug and snai1 in A549 and H1299 cells compared with the scramble control. Reverse results were observed in A549 cells with ectopic overexpression of

DSTN (Fig. 6B), and the optical density of each protein was quantified by GAPDH optical density.

Moreover, the above results were further confirmed by real-time PCR using GAPDH as an internal control (Fig. 6C-E). These results suggest that the decrease in c-Myc, cyclin D1, slug and snai1 occurred after inhibition of the Wnt/β-catenin pathway when blocking DSTN in NSCLC cells. Our results indicate that overexpressed DSTN could be a critical stimulating factor for the activation of the Wnt/β-catenin pathway in NSCLC cells.

DSTN overexpression promotes β-catenin nuclear translocation and enhances its interaction with TCF4

In most cases, the elevated expression of β-catenin is the cause of activating the canonical pathway.

In the canonical pathway, β-catenin combined with LEF/TCF4 complex in the nucleus to activate

Wnt signaling (32, 33). Accordingly, to gain insight into the underlying mechanisms through which DSTN knockdown inhibits Wnt/β-catenin signaling, we first examined expression and nucleus subcellular localization of β-catenin in DSTN knockdown and control cells using immunofluorescent staining and Western blot analysis. Our results revealed that DSTN knockdown did not cause the significant changes of β-catenin expression level in A549 and H1299 cells compared with shNC control cells but led to abnormal distribution of β-catenin in cells. As shown in suppl. Fig. 12, localization of β-catenin was mainly in the cytoplasmic membrane of

A549 and H1299 cells when blocking DSTN by Immunofluorescent staining. Similarly, Western blot analysis further showed a decrease of β-catenin proteins in nuclear fractions of A549 and

H1299 cells, which stably infected with shDSTN (Fig. 7A). Consistently, ectopic overexpression of DSTN did not affect the protein level of β-catenin expression as well but caused β-catenin to mainly localize in the nucleus in A549 cells with DSTN overexpression compared with vector control. Furthermore, the lentiviral DSTN-expressing vector in DSTN knockdown cells efficiently restored nucleus subcellular localization of β-catenin. These data thus far promoted us to ask whether DSTN interacts with β-catenin in NSCLC cells. To test this possibility, we conducted reciprocal immunoprecipitation experiments. We observed that DSTN and β-catenin interact with each other in HEK293T cells co-expressing β-catenin and DSTN (Suppl. Fig. 13), and interaction of endogenous β-catenin and DSTN in A549 and H1299 cells (Fig. 7B), suggesting that DSTN 14

could be an important factor to enhance β-catenin nuclear localization. Additionally, we wonder whether DSTN expression affects the interaction of β-catenin with TCF4. We thus further examined the interaction of β-catenin and TCF4 in A549 and H1299 cells stably infected with shDSTN or scramble control. Immunoprecipitation results showed that knocking down DSTN expression in A549 and H1299 cells markedly impaired interaction of β-catenin and TCF4, whereas the interaction of β-catenin and TCF4 was increased in A549 cells with ectopic expression of DSTN (Fig. 7C). Rescue experiment showed that the DSTN-expressing vector efficiently restored the interaction of β-catenin and TCF4 in A549 and H1299 with DSTN knockdown. Taken together, the present study additionally identified the regulatory role of DSTN as an activator of the Wnt/β-catenin signaling pathway, and demonstrated that alteration in DSTN expression led to an interchange of localization pattern of β-catenin between the nucleus and cytoplasmic membrane in A549 and H1299 cells.

DISCUSSION

In the present study, we provided strong evidence supporting that enforced expression of DSTN is a crucial aggravating factor of LUAD progression and metastasis. DSTN expression is vital in facilitating LUAD cell proliferation and invasion through its coordinated augment of β-catenin nuclear trans-localization, leading to its interaction with TCF4. Overexpression of DSTN correlates with EMT, lymph node metastasis, late-stage LUAD and poor survival of LUAD. In addition, due to its aberrant expression level in LUAD, our results suggest that DSTN can serve as an initiation factor and/or a prognostic biomarker of LUAD metastasis.

Previous studies have demonstrated that actin-binding proteins (ABPs), which are responsible for polymerization and treadmilling of actin, regulate actin filament dynamics (34-36). One of the most important ABP families is the actin-depolymerizing factor (ADF)/cofilin family, which includes destrin (DSTN), cofilin-1 and cofilin-2 (37, 38). These proteins are the key regulators in regulating actin cytoskeleton remodeling, a crucial process for cytokinesis, as well as regulating in polarization involved in cell growth, migration and invasion of cancer cells, but their property of promoting proliferation and metastasis has not yet been reported in human LUAD. Especially,

DSTN is far less characterized in this family than other numbers; Moreover, it remained unknown whether DSTN involved in epithelial-mesenchymal transition (EMT) and/or how it aggravates 15

LUAD progression.

We found a dramatic elevation of DSTN in tumor tissues of the mouse lung cancer model compared with control mice using iTRAQ analysis. Consistent with this, a frequently up-regulation of DSTN expression in human LUAD tissues was identified. Simultaneously, we observed that DSTN upregulation was associated with the progression and liver metastasis of lung cancer in the mouse model. Moreover, high DSTN expression in tumors was significantly correlated with lymph node metastasis of LUAD, clinical TMN stage and worse overall survival of clinical LUAD patients. In lines with our findings, up-regulated DSTN has been found in pancreatic cancer as well (16). Conversely，DSTN knockdown markedly inhibits migration and invasion of human colon cancer Isreco1 cells (15). These data add support to our animal model data, clinical and cellular model data, suggesting that DSTN may play a critical role in the initiation and/or progression of LUAD. Moreover, it may serve as a potential therapeutic/gene therapy target, as well as a biomarker to predict the progression and prognosis of LUAD in the future.

DSTN (also known as an actin-depolymerization factor, ADF) is an essential actin regulatory protein of the ADF/cofilin family. Mechanically, it binds to the subunits of filamentous actin, enhances the subunit off-rate and promotes filaments severing, which is required for tumor cell migration and invasion (11, 12). However, the mechanism by which DSTN contributes to the tumor has been still poorly understood. Experimental and clinical studies have shown that the

EMT process plays a gatekeeper role in tumor cells invasion, metastasis and even chemoresistance

(39, 40). Overexpression DSTN enhances NSCLC cell migration and invasion probably through the EMT, because DSTN knockdown in NSCLC cells results in lowered levels of Vimentin and

N-cadherin but an increase of E-cadherin and γ-catenin levels. However, overexpression of DSNT in A549 reduced E-cadherin and γ-catenin expression and increased levels of Vimentin and

N-cadherin expression, indicating a positive correlation between DSTN and EMT, which might account for the potential invasion and metastasis of those cells with a high level of DSTN. In addition, our results also showed that overexpressed DSTN in A549 cells increased slug, snai1 mRNA and protein levels (important transcriptional repressor of E-cadherin and inducer of EMT), while DSTN knockdown markedly decreased slug and snai1 expression levels. It has been well-known that E-cadherin and Vimentin are the major regulators involved in the EMT process. 16

Moreover, E-cadherin down-regulation and/or Vimentin up-regulation are cornerstones for the initiation of EMT, the gain of invasion and installation of metastasis of cancer cells (41, 42). We speculated that DSTN-dependent expression of EMT relevant proteins might be a potential mechanism, by which they lead to LUAD metastasis.

It is well established that a variety of signaling pathways is involved in the activation of EMT programs, including Wnt/β-catenin signaling (43). However, the physical and biological relationships between EMT and the Wnt/β-catenin pathway have not been thoroughly understood.

Wnt/β-catenin signaling is tightly regulated at multiple cellular levels and is dysregulated in numerous types of cancer, including lung cancer (44). Abnormal activation of the Wnt/β-catenin pathway is a crucial oncogenic step to tumor initiation and progression (45). β-catenin is a key component of Wnt/β-catenin signaling. Moreover, it is a dual-function protein that is involved in transcriptional regulation and cell-cell adhesion. In normal epithelial cells, β-catenin interacts with

E-cadherin to form an adhesion complex, which locates in cell-cell adherent junctions at the membrane. In tumor cells, activated β-catenin can translocate to the nucleus and interacts with

TCF/LEF transcription factors to activate transcription of Wnt targeting genes. In addition, it has been reported that EMT also induces a switch from the β-catenin/E-cadherin complex to the

β-catenin/ TCF4/Twist1 complex, which then binds to cancer stem cell-related gene promoters to activate oncogene expression (46). Our studies show that silencing DSTN promotes apoptosis and induces G2/M arrest in NSCLC cells. Moreover, DSTN knockdown could induce the abnormal intracellular distribution of β-catenin and the expression dysregulation of multiple downstream genes of Wnt/β-catenin, such as c-myc, cyclin D1, slug and snai1 in A549 and H1299 cells.

Immunofluorescence assay showed that β-catenin is mainly anchored on the cytoplasmic membrane and its nuclear distribution was significantly decreased in DSTN-knockdown cells

(Fig.7A). More interestingly, a Co-immunoprecipitation assay of the nuclear fraction revealed that

DSTN is associated with β-catenin in the nucleus. We found that this interaction increased the translocation ability of β-catenin to the nucleus in A549 cells with the enforced expression of

DSTN. Moreover, further analysis confirmed that DSTN depletion affects the interaction of

β-catenin with TCF4, leading to a decrease in the complex formation of β-catenin -TCF4 transcription factor in NSCLC cells. These results promote us to postulate that DSTN overexpression contributes to nuclear entry of β-catenin and the formation of β-catenin-TCF4 17

transcription factor complex; and the latter is an important process in the development of NSCLC

metastatic potential and EMT phenotype (47, 48). Therefore, our findings provide new insights

into the roles of DSTN that facilitate invasion and metastasis of LUAD, suggesting DSTN may

promote NSCLC cell proliferation and metastasis by activating Wnt/β-catenin signaling pathway.

In summary, we not only provide evidence that DSTN participated in lung adenocarcinoma

development and progression, and its overexpression indicated a poor survival of LUAD patients,

but also illustrated the critical role of the interaction between DSTN and β-catenin, by which EMT

is established in cancer metastasis. Collectively, we showed the importance of DSTN in lung

adenocarcinoma malignancy and proposed that DSTN is a potential therapeutic target and a

biomarker for LUAD. The proposed DSTN-β-catenin axis may offer a new avenue to develop

novel approaches anti-LAUD in the future.

Acknowledgements

This work was supported by National Natural Science Foundation of China (No. 81372147) and

Henan University support grant CX3070A0780502

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Figure Legends Figure 1. characterization of DSNT expression in lungs following urethane treatment. (A-D)

Representative images of immunohistochemical staining for DSTN in the lungs of control mice (A) and the mice at week 20 (B), 28 (C), and 32 (D) after urethane treatment. (E) The number of cells expressing DSTN was evaluated by immunohistochemistry 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=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

32w post-treatment, 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, 100μm. (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. the below panel is the quantified results by Grayscale scanning. *p

＜0.05, **p＜0.01.

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 non-tumor tissues (N). GAPDH was used as loading control in the immunoblotting assay. The upper panel is the signal intensity of DSTN and GAPDH; the lower panel is the quantified results by Grayscale scanning. n=20, * p＜0.05. (B) Real-time PCR analysis of DSTN mRNA levels in 20 paired lung adenocarcinoma tissues (T) and adjacent non-tumor tissues (N). The expression of GADPH was used as loading control in real-time PCR assays. ** p＜0.01，*** p＜0.001. (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 non-tumor tissues (N). All tissue samples used in this experiment have the same or similar clinicopathological characteristics except for the status of lymph node metastasis. The right panel is the quantified results by Grayscale scanning. n=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 clinicopathological characteristics except for clinical TNM stages. The right panel is the quantified results by

Grayscale scanning. n=9, *p ＜ 0.05. (E) Representative positive and negative immunohistochemical staining of DSTN in lung adenocarcinoma tissues (Ⅰ, Ⅱ) and their adjacent non-tumor tissues (Ⅲ, Ⅳ) (magnification, ×200). (F, G) Kaplan-Meier analysis of overall survival of lung adenocarcinoma patients 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.

Figure 3. The effect of DSTN on the proliferation of LUAD cells in vitro and in vivo. (A) DSTN expression level in BEAS-2B and five human NSCLC cell lines was analyzed by Western blot. (B)

DSTN silencing efficiency of sh1DSTN, sh2DSTN and sh3DSTN in A549 and H1299 cell lines was assessed respectively by Western blot analysis. (C) Downregulated or upregulated DSTN did not affect the expression of a cofilin-1 protein in A549 and H1299 cells. (D) The effect of DSTN knockdown and overexpression on cell growth was evaluated by MTT assay in A549 and H1299 cells, respectively. (E) The effect of DSTN knockdown or overexpression on colony formation in

A549 and H1299 cells. The data were present as mean ± SD values from three independent experiments. Asterisks (*) indicate statistical significance: **p＜0.01, ***p＜0.001. (F) in vivo

tumor formation assay of 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 was inhibited by DSTN knockdown. n=5, *p＜0.05,

***p＜0.001.

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, 200μm). 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=5, ***p＜0.001.

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,100μm). (B) The effect of DSTN knockdown or overexpression on EMT marker expression, including E-cadherin, N-cadherin,

Vimentin, and γ-catenin, was assessed by Western blot. 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, 200μm). Statistical data shown is the mean ± SD of three independent assays. Asterisks (*) indicate statistical significance: ***p＜0.001.

Figure 6. DSTN knockdown or overexpression affects Wnt target gene expression by regulating activity of the Wnt/β-catenin signaling pathway. (A) The effect of DSTN knockdown or overexpression in A549 and H1299 cells on the Wnt/β-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 of DSTN knockdown in A549 and H1299 cells and overexpression in A549 on mRNA 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.

Figure 7. DSTN knockdown or overexpression affects the sub-cellular localization of β-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 β-catenin in A549 and H1299 cells was assessed by Western blot analysis. (B) Co-immunoprecipitation showed an interaction between endogenous β-catenin and DSTN in A549 and H1299 cells. Cell lysate was incubated with an anti-β-catenin or anti-DSTN antibody for the IP experiment. IgG was used as a negative control.

DSTN knockdown inhibited the interaction of endogenous β-catenin and TCF4 in A549 and

H1299 cells, whereas DSTN overexpression increased the interaction of endogenous β-catenin and

TCF4 in A549 cells.

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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