Overexpression of RCC2 Enhances Cell Motility and Promotes Tumor Metastasis in Lung Adenocarcinoma by Inducing Epithelial-Mesenchymal Transition

Author Manuscript Published OnlineFirst on June 12, 2017; DOI: 10.1158/1078-0432.CCR-16-2909 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Bo Pang1,#, Nan Wu1,#, Rongwei Guan1,#, Lin Pang3, Xinlei Li4, Su Li1, Liudi Tang1, Ying Guo1, Jialei Chen5, Donglin Sun1, Haiming Sun1, Jialin Dai1, Jing Bai1, Guohua Ji1, Peng Liu1, An Liu1, Qiushi Wang5, Sheng Xiao6, Songbin Fu1,2,*, Yan Jin1,2,*

1Laboratory of Medical Genetics, Harbin Medical University, Harbin 150081, China 2Key Laboratory of Medical Genetics (Harbin Medical University), Heilongjiang Higher Education Institutions, Harbin 150081, China 3College of Bioinformatics Science and Technology, Harbin Medical University, Harbin 150081, China 4Department of human anatomy, Harbin Medical University, Harbin 150081, China 5Department of thoracic surgery, The Second Affiliated Clinical Hospital, Harbin Medical University, Harbin 150081, China 6Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA

*To whom correspondence should be addressed.

#These authors contributed equally to this work.

Running title: RCC2 Promotes Lung Adenocarcinoma Metastasis

Keywords: RCC2, lung adenocarcinoma, metastasis, EMT

*Correspondence: Dr. Songbin Fu (E-mail: [email protected], [email protected]) or Dr. Yan Jin (E-mail: [email protected]), Laboratory of Medical Genetics, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin 150081, People’s Republic of China, Tel: 86–451-86674798, Fax: 86–451-86674798.

Grant Support: This work was supported by the National Natural Science Foundation of China (Grant No. 81372784 for Songbin Fu, [email protected] and Grant No. 81371617 for Yan Jin, [email protected]), the Program for Changjiang Scholars and Innovative Research Team in University of China (Grant No. IRT1230 for Yan Jin, [email protected]), and the National Key Research and Development Program of China (Grant No. 2016YFC1000504 for Songbin Fu, [email protected]), the Outstanding Youth foundation of Heilongjiang Province of China (Grant No. JC201215 for Yan Jin, [email protected]).

Conflict of interest: The authors declare no potential conflicts of interest.

Word count: 5111 Number of Tables: 2 Number of Figures: 6

This manuscript has not been presented previously.

Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 12, 2017; DOI: 10.1158/1078-0432.CCR-16-2909 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Translational Relevance

A high incidence of tumor recurrence and metastasis has been reported in LUAD patients. However, the underlying mechanisms are poorly understood. To reduce LUAD-related metastasis, diagnosis of the disease at early stage is highly desirable. In this study, we found RCC2 was frequently overexpressed in LUADs, which was significantly associated with metastasis and advanced clinical stage. LUAD patients with RCC2 overexpression had poorer overall survival and RCC2 also determined to be an independent prognostic factor for poorer outcome in LUAD patients. Moreover, a series of in vitro and in vivo studies demonstrated that RCC2 overexpression obviously promoted LUAD metastasis by inducing EMT. Our findings suggested that RCC2 may serve as a new prognostic biomarker for predicting patient outcome and as a potential therapeutic target for improving the efficacy of accurate treatment.

Abstract

Purpose: Investigate the role of regulator of chromosome condensation 2 (RCC2) on lung adenocarcinoma (LUAD) metastasis.

Experimental Design: Clinical specimens were used to assess the impact of RCC2 on LUAD metastasis. Mouse models, cytobiology and molecular biology assays were performed to elucidate the function and underlying mechanisms of RCC2 in LUAD.

Results: RCC2 expression was frequently increased in LUADs (88/122, 72.13%). It was confirmed by analysis of a larger cohort of TCGA RNA-seq data containing 488 LUADs and 58 normal lung tissues (P<0.001). Importantly, increased level of RCC2 was significantly associated with T status of tumor (P =0.002), lymph node metastasis (P =0.004) and advanced clinical stage (P =0.001). LUAD patients with higher expression of RCC2 had shorter overall survival. Cox regression analysis demonstrated that RCC2 was an independent poorer prognostic factor for LUAD patients. Moreover, forced expression of RCC2 promoted intra-pulmonary metastasis in vivo and significantly enhanced LUAD cell migration, invasion and proliferation in vitro. Further study found that RCC2 induced epithelial-mesenchymal transition (EMT) and also stimulated the expression of MMP-2 and MMP-9. In addition, RCC2 was able to activate JNK, while inhibition of JNK suppressed the effect of RCC2 on LUAD cell migration, invasion, EMT and the expression of MMP-2 and MMP-9.

Conclusion: RCC2 plays a pivotal role in LUAD metastasis by inducing EMT via activation of MAPK-JNK signaling.

Introduction

Lung cancer, the foremost cause of global cancer-related mortality(1), is responsible for over a million deaths each year worldwide, with adenocarcinoma being the most common histological type(2). Lung adenocarcinoma (LUAD) comprises ~40% of all lung cancer with increasing incidence in both Asian and Western countries(3). Notably, the average 5 year survival rate for LUAD is about 15%(4) and has not improved in the past few decades(5), mostly since patients eventually succumb to metastatic relapse after surgical resection, who are almost always incurable(6). Tumor metastasis is the major cause of LUAD-related death, moreover, somatic mutations of many oncogenes and perturbation of key pathways have been frequently discovered in LUAD(7). Therefore, it is strongly needed to reveal the molecular mechanisms underlying LUAD genesis and development and to identify effective prognosis biomarkers for high-risk patients with LUAD.

Regulator of chromosome condensation 2 (RCC2), also known as TD-60, was originally identified using human autoimmune antiserum at the spindle midzone in anaphase and telophase(8). RCC2 was reported as a component of the chromosomal passenger complex (CPC) together with Aurora B kinase(9), INCENP(10) and Survivin(11) involving in regulating chromosomes and the spindle assembly in mitosis and cytokinesis process(12, 13). And recent studies showed that RCC2 was associated with the integrin adhesion complexes and involved in cell migration during interphase, suggesting its role in cell signaling and interphase cell cycle progression(14-17). Growing evidence indicated that RCC2 was related to RCC1 (regulator of chromatin condensation 1), a guanine exchange factor (GEF) for Ran and also exhibited GEF activity for the small GTPases Rac1(12) and RalA(13). The gene RCC2 was also reported to play a potential role in tumorigenesis. Matsuo M et al. showed that RCC2, as the target of tumor-suppressor miR-29c, was upregulated in gastric carcinoma tissues and contributed to the proliferation of gastric carcinoma cells by regulating cell cycle progression(18). Two genome-wide SNP association analysis showed the SNP rs7538876, which mapped in the vicinity of RCC2, conferred risk of melanoma(19) and cutaneous basal cell carcinoma (BCC)(20) progression. And a series of experiments confirmed the potential association of rs7538876 with the expression of RCC2. Feng N et al. found that ENST00000439577, a long non-coding RNA (lncRNA), was positively correlated with the expression of RCC2. Furthermore, high expression of ENST00000439577 was associated with metastasis status and poor overall survival in non-small cell lung cancer (NSCLC)(21). Though Bruun J et al. demonstrated that the 5’UTR mutation of RCC2 reduced protein expression, and was significantly associated with poor prognosis in microsatellite stable (MSS) tumors(22). However, the potential correlation between RCC2 and LUAD progression is still unclear.

Herein, we reported that overexpression of RCC2 in LUAD was important in the acquisition of an aggressive and poor prognostic phenotype. Overexpression of RCC2 promoted LUAD cell proliferation and metastatic capacity both in vitro and in vivo. More importantly, we demonstrated, for the first time, that RCC2 regulated LUAD cell epithelial-mesenchymal transition (EMT) and extracellular matrix remodeling via JNK activation, ultimately driving the aggressiveness of cancer cells. Our results provided functional and mechanistic links between RCC2 and LUAD.

Materials and Methods

Tissue samples and TMAs

Twelve fresh tissues including LUAD (n=6) and paired adjacent normal lung (n=6) were obtained from the Second Affiliated Clinical Hospital (Harbin Medical University, Harbin, China). No patient had received chemotherapy or radiotherapy before surgery. Samples used in this study were approved by the Institutional Review Board of Harbin Medical University (HMUIRB20160025). All procedures were conducted in accordance with the Declaration of Helsinki and International Ethical Guidelines for Biomedical Research Involving Human Subjects (CIOMS). Written informed consents were obtained from all patients. In addition, the tissue microarrays (TMAs) consisted of 122 pairs of human LUAD and their adjacent non-neoplastic lung tissues and TMA containing 120 human LUAD tissues were purchased from Outdo Biotech CO. Ltd. (Shanghai, China).

Data acquisition

We download the RNA-seq data (level 3) and corresponding clinical information of 488 patients with lung adenocarcinoma and 58 normal tissues from The Cancer Genome Atlas (TCGA) data portal (https://tcga-data.nci.nih.gov/tcga/tcgaDownload.jsp). The expression levels for each gene were calculated as TPM values, which were defined by RSEM(23).

Cell lines and cell culture

Human LUAD cell lines A549, NCI-H1650, and NCI-H23 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Other LUAD cell lines including NCI-H1975 and HCC827 were purchased from the Xiangf Biological Technology Co. Ltd. (Shanghai, China). A549 cell lines were routinely cultured in Ham’s F12K media. NCI-H1650, NCI-H1975, HCC827 and NCI-H23 were cultured in Roswell Park Memorial Institute (RPMI) 1640 media. All cell lines were routinely cultured in media supplemented with 10% fetal bovine serum (PAA Laboraties GmbH, Pasching, Austria) at 37°C in a humidified atmosphere containing 5% CO2.

Overexpression and RNA interference of RCC2

A549 and NCI-H1650 cells were transfected with RCC2-eYFP by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. RCC2 construct were kindly gifted from Dr. Sheng Xiao (Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, USA). For the establishment of the A549-RCC2 cell line stably expressing RCC2, 24h after transfection, cells were maintained in Ham’s F12K media containing 1000 μg/mL of G418 (Calbiochem, Darmstadt, Germany). After three weeks, resistant colonies stably transfected with RCC2 were pooled. Short interfering RNAs (siRNAs) specifically against RCC2 and corresponding scrambled siRNA were purchased from RiboBio (Guangzhou, China) and then transiently transfected into A549-RCC2 and HCC827 cell lines using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. The gene silencing effect was measured by western blotting 48h post-transfection.

Western blotting

Western blot analysis was performed according to the standard protocol. Proteins were extracted from cells and tissues with RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) complemented with protease inhibitors (Roche). For western blot analysis, protein extracts were subjected to electrophoresis on 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Immunoblots were blocked with 5% BSA in TBS/Tween 20 and incubated with primary antibodies (Supplementary Table 1) overnight at 4°C. The bands were visualized by the ChemiDoc TM MP Imaging System (BIO-RAD). The specificity of anti-RCC2 antibody used in this study was verified according to the proposal for validation of antibody (24) (Supplementary Fig. 1).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted using the High Pure RNA Isolation Kit (Roche, Basel-Stadt, Switzerland) and reverse transcription was performed with the PrimeScript ® RT Reagent Kit Perfect Real Time (Takara, Dalian, China). The cDNA was subjected to quantitative RT-PCR using the LightCycler ® 480 SYBR Green I Master (Roche) and the assay was performed on a CFX96TM Real-Time System (BIO-RAD) following the manufacturer’s instructions. GAPDH was used to normalize the amount of cDNA between different samples. Primer sequences were listed as follows: RCC2 forward 5′-TTCCTTTGGGTGCCCTGAA-3′, reverse 5′-GGCAGAATCTGTCCATCTTTCG-3′, GAPDH forward 5′-ATCACTGCCACCCAGAAGAC-3′, reverse 5′-TTTCTAGACGGCAGGTCAGG-3′.

Proliferation assay

Cells were seeded in triplicate in 24-well plates. Cell numbers were counted every 24h after cell adhesion over a 5 day period. The experiment was independently repeated three times.

Wound-healing assay

For the wound-healing assay, cells were grown on a six-well plate until 95% confluence and monolayers were scratched with a pipette tip. Cell migration was recorded at 0h, 24h, and 48h after wound scratch. Three independent experiments were performed.

Cell invasion and migration assays

Invasion and migration assays were performed using Corning chambers (Corning, MA, USA) with or without Matrigel following the manufacturer’s protocol. Briefly, cell suspensions prepared in media containing 2% FBS were added into upper chambers, while media containing 20% FBS was placed in the lower chambers as a chemoattractant stimulus. The cells were incubated for 24h or 48h at 37℃ and allowed to migrate or invade through the membrane filter. Afterward, the non-migratory and non-invasive cells on the upper surface were gently removed by a cotton swab. Then, cells that had invaded or migrated to the lower surface of the membrane were fixed with methanol and stained with hematoxylin and eosin. Cells in four randomly microscopic fields (at 100× magnification) were counted and photographed. Both experiments were independently repeated in triplicate.

Immunocytochemistry (ICC) analysis

Cells were transfected with RCC2-eYFP and cultured in media. After fixing with 4% paraformaldehyde (PFA) for 15 minutes, cells were then permeabilized with 0.5% Triton x-100 for 20 min. Primary antibody against RCC2 (1:100) was incubated overnight at 4°C. Sequentially, the

cells were incubated with secondary antibodies anti-rabbit at 37°C for 30 min. DAB-H2O2 was used for the color reaction and all of the cells were counterstained with hematoxylin.

Immunohistochemistry (IHC) analysis

Immunohistochemical (IHC) staining was performed using PowerVision™ Two-Step Histostaining Reagent (Zhongshan Golden Bridge, Beijing, China) according to the manufacturer’s instructions. After deparaffinization and rehydration, tissue slides were routinely

treated with 3% H2O2 at room temperature for 10 min. 10 mM EDTA was used for antigen retrieval. Then rabbit primary antibodies against human RCC2 (1:100 for TMAs, 1:400 for mouse tissues), N-cadherin (1:100 for TMAs), α-SMA (1:200 for TMAs), β-catenin (1:200 for TMAs), E-cadherin (1:200 for TMAs) and p-JNK (1:50 for TMAs) were added to the tissues and incubated overnight at 4°C in a moist chamber. Sequentially, the slides were incubated with goat anti-rabbit

IgG at 37°C for 30 min. After washing in PBS, DAB-H2O2 was used for the color reaction. Finally, all of the slides were counterstained with hematoxylin, dehydrated and mounted. For negative controls, the processing was performed as described above, except that they were incubated overnight without the primary antibody at 4°C. Immunoreactivity on TMAs was assessed independently by three researchers. The specificity of anti-RCC2 antibody was verified according to the proposal for validation of antibody (24) (Supplementary Fig. 1).

Immunofluorescence analysis

Cells were fixed with cold ethanol for 20 min and permeabilized with 0.1% Triton x-100 for 5 min. The samples were washed with PBS and blocked with 5% BSA in PBS. Subsequently, cells were incubated with primary antibodies against β-catenin, E-cadherin, N- cadherin (Proteintech, 1: 50 dilution) overnight at 4°C and then incubated with fluorescence-labeled rabbit antibody. The nuclei were counterstained with 4’-6-diamidino-2-phenylindole (DAPI) for 5 min.

Xenograft formation and in vivo metastasis assay

All animal experiments were conducted in accordance with the ethical standards and national guidelines and approved by the Institutional Review Board of Harbin Medical University (HMUIRB20160025). Female BALB/c-nude mice (4-5 weeks old), purchased from Vital River Laboratory (Beijing, China), were kept under standard conditions. Xenograft tumor growth assay was established by subcutaneous injection of A549-RCC2 cells and control cells (7×106, suspended in PBS) into the right dorsal flank. To preparation of xenograft model for RCC2 knockdown study on tumor growth, HCC827 cells (1× 107) were subcutaneously injected into the right ﬂanks (four mice per group). Ten days after tumor inoculation, cholesterol-conjugated RCC2 siRNAs (siRNA1 and siRNA2) and their negative controls were from RiboBio (Guangzhou, China) for in vivo siRNA delivery. 10 nmol siRNA in 0.1 ml saline buffer was injected into tumor mass once every 3 days for 3 weeks(25, 26). Tumor volume (TV) was measured by calipers and calculated as formula: TV (mm3) = (L × W2)/2 (L, long diameter; W, wide diameter). After sacrifice, tumors were excised for following assays. For the intravenous mouse model, two groups

of four mice each were given intravenous tail vein injections of 6×106 A549-RCC2 cells and control cells, respectively. After 8 weeks the mice were sacrificed and the tumor nodules formed on the lung surfaces were counted. Lungs were collected and embedded in paraffin for further hematoxylin and eosin (H&E) staining and IHC analysis.

Statistical analysis

All statistical analysis were performed using R software (version 3.2.2) (http://www.R-project.org/) or SPSS 17.0 (IBM; Armonk, NY, USA). The differential expression level of RCC2 between LUAD and adjacent normal tissues was evaluated by McNemar’s chi-squared test. The correlation between RCC2 expression and clinicopathologic features of LUAD patients was analyzed by the χ2 test or Fisher’s exact test. Survival curves were obtained using the Kaplan-Meier method, and statistical analysis was performed using the log-rank test. Cox proportional hazards regression model was performed for univariate and multivariate survival analyses. Other data were expressed as the means ± standard deviation (SD), and the independent Student’s t test was applied to analyze the statistical significance between two groups. P<0.05 was considered statistically significant.

Results

RCC2 overexpression was frequently identified in LUAD and associated with tumor metastasis and poor prognosis of patients

To investigate the expression status of RCC2 in LUAD, tissue microarrays (TMAs) which contained 122 pairs of LUAD specimens and corresponding adjacent noncancerous tissues and detailed pathological information with survival prognosis of patients were examined by immunohistochemical (IHC) staining. The frequency of positive staining of RCC2 was significantly higher in tumors (88 of 122 [72.13%]) than that in nontumor tissues (11 of 122 [9.02%]) (P<0.001). IHC staining of RCC2 in representative samples of LUAD and normal lung tissues was shown in Fig. 1A-B. To further confirm our observation, we expanded the analysis to a larger cohort of TCGA RNA-seq expression data that contained 488 lung adenocarcinoma and 58 normal lung tissues. Again, RCC2 expression in adenocarcinoma was statistically (P<0.001) higher than that in the normal tissues (Fig. 1C). In addition, RCC2 protein expression between 6 pairs of fresh LUAD tissues (T) with their adjacent normal lung tissues (N) was compared by western blotting. As shown in Fig. 1D, the result revealed a similar trend that RCC2 overexpression was detected in 4/6 (66.67%) of LUAD tissues compared with the matched nontumor tissues.

We next evaluated the association of RCC2 overexpression with clinicopathologic features in 122 LUAD samples with informative IHC data. The results showed that RCC2 overexpression was significantly associated with T status of tumor (P=0.002), lymph node metastasis (P=0.004) and a more advanced clinical stage (P=0.001, Table 1). Kaplan-Meier survival analysis showed that LUAD patients with higher expression of RCC2 had significantly poorer overall survival using TMA data (P=0.003, log-rank test, Fig. 1E), TCGA dataset (P=0.032, log-rank test, Fig. 1F) of LUAD and the Gene Expression Omnibus (GEO) dataset (GSE31210) from PrognoScan (P=0.002, log-rank test, Supplementary Fig. 2). Further multivariate Cox regression analysis demonstrated that RCC2 overexpression was an independent prognostic factor for poor survival of

patients with LUAD in TCGA dataset (HR, 1.687; 95% CI 1.098 to 2.594; P=0.017, Table 2), though there was no significance in TMA data (Supplementary Table 2). In conclusion, these findings suggested that overexpression of RCC2 was frequently detected in LUAD and its overexpression was associated with tumor metastasis and poor prognosis in LUAD patients.

Overexpression of RCC2 promoted proliferation, migration and invasion of LUAD cells

To explore the impact of RCC2 on the biological characteristics of LUAD cells, we first assessed expression levels of endogenous RCC2 in a panel of 5 LUAD cell lines (A549, NCI-H1650, HCC827, NCI-H1975, NCI-H23) by western blotting (Fig. 2A). A549 and NCI-H1650 cell lines showed low endogenous RCC2 expression in this group and therefore were selected for an overexpression study. A RCC2 expression construct was transfected into A549 and NCI-H1650 cells, and then the expression of exogenous RCC2 was demonstrated by western blotting (Fig. 2B). These cells were subjected to a cell growth assay, which showed significantly increased cell proliferation in RCC2-transfected cells as compared to the control cells (Fig. 2C). Because our clinical correlation analysis revealed association of RCC2 overexpression with LUAD metastasis, we investigated the impact of RCC2 on LUAD cell motility and invasiveness. Wound-healing assay showed that overexpression of RCC2 enhanced A549 cell migration at the edge of exposed regions (Fig. 2D). Further, migration and Matrigel invasion assays in vitro were also performed in A549 and NCI-H1650 cells. The results showed that the expression of exogenous RCC2 in LUAD cells significantly increased cell motility (P<0.001, Fig. 2E-F) and invasion potential (P<0.001, Fig. 2G-H) as compared to control cells. These results indicated that RCC2 overexpression enhanced LUAD cell proliferation, migration and invasion in vitro.

Inhibition of RCC2 suppressed LUAD cell proliferation, migration and invasion

To further confirm whether RCC2 silencing could inhibit cell growth and invasiveness, the RCC2 gene was silenced by RNA interference (RNAi) using two targeted siRNAs (siRNA1 and siRNA2) in A549 cells with stable RCC2 overexpression and HCC827 cells with relatively high endogenous RCC2 expression. Both siRNAs efficiently knocked down RCC2 in LUAD cells (Fig. 3A). RCC2 depletion led to decrease cell growth (Fig. 3B) and wound healing ability as compared to control cells (Fig. 3C-D). Migration and Matrigel invasion assays also demonstrated that ablation of RCC2 markedly reduced the migration and invasion capacity of both A549-RCC2 and HCC827 cell lines (P<0.01, Fig. 3E-F). Collectively, these results provided evidence that RCC2 silencing could inhibit LUAD cell tumorigenicity in vitro.

RCC2 induced epithelial-mesenchymal transition (EMT) and extracellular matrix remodeling in LUAD

Since epithelial-mesenchymal transition (EMT) and extracellular matrix remodeling are the key events in driving tumor invasion and metastasis. To investigate the effects of RCC2 on EMT and extracellular matrix remodeling, we analyzed the expression levels of EMT markers, EMT-related transcription factors and matrix metalloproteinases (MMPs). RCC2-transfected A549 and NCI-H1650 cells showed increased expression of mesenchymal markers (N-cadherin and α-SMA), EMT-related transcription factors (Snail and Slug), and two MMPs (MMP-2 and MMP-9), while the expression of β-catenin and E-cadherin was decreased (Fig. 4A). Immunofluorescent staining assays confirmed the increased expression of N-cadherin and

decreased expression of β-catenin in the A549-RCC2 cells (Fig. 4C). On the contrary, after the silence of RCC2 in A549-RCC2 and HCC827 cells, the levels of β-catenin and E-cadherin were upregulated, whereas the levels of N-cadherin, α-SMA, Snail, Slug, MMP-2 and MMP-9 were downregulated obviously (Fig. 4B). To determine the clinical relevance, TMAs containing 120 human LUAD specimens (119 specimens available) were subjected to IHC staining for RCC2 and EMT markers (N-cadherin, α-SMA, β-catenin, and E-cadherin) in serial sections (Supplementary Fig. 3). Consistently, IHC analysis confirmed that RCC2 expression level was positively correlated with the expression levels of N-cadherin and α-SMA, and inversely correlated with the expression levels of β-catenin and E-cadherin (Supplementary Table 3). The correlations of the RCC2 expression level with N-cadherin, Snail, MMP-2 and MMP-9 were further studied in the same cohort of LUAD samples of TCGA dataset. The results confirmed that RCC2 expression was significantly correlated with the expression of N-cadherin (R=0.284, P<0.001), Snail (R=0.238, P<0.001) MMP-2 (R=0.171, P<0.001), and MMP-9 (R=0.176, P<0.001) (Fig. 4D). Taken together, these results suggested that the RCC2 is associated with EMT and extracellular matrix remodeling, which likely contribute to tumor metastasis in LUAD.

RCC2 promoted xenograft tumor growth and metastatic potential in mouse models

To assess whether overexpression of RCC2 could enhance the tumorigenic capacity in vivo, the xenograft tumor mouse model was established by subcutaneously injecting A549-Vec and A549-RCC2 into the right dorsal flanks of nude mice. During the course of nine weeks, tumor volume was monitored by a caliper, which showed significantly larger tumors in the group with RCC2 overexpression as compared to control mice (P<0.05, Fig. 3A). At the end of experiments, xenograft tumors were isolated and the weights were measured, which again showed increased tumor size and weight in mice with RCC2 overexpression (Fig. 3B-C). Moreover, these tumors appeared multinodular in A549-RCC2 group compared with the control group. Results from the hematoxylin and eosin (H&E) staining confirmed the tumor formation (Fig. 3D). Further, the expression of RCC2 in xenograft tumors developed from the RCC2-transfected cells was confirmed by IHC staining and qRT-PCR (Fig. 3D-E). We further verified several EMT markers (N-cadherin and E-cadherin) by western blotting using tumor tissues from xenograft mice. Compared with the A549-Vec group, N-cadherin protein level significantly increased while the expression of E-cadherin decreased in A549-RCC2 group (Fig. 3F).

To demonstrate whether RCC2 silencing could inhibit tumorigenicity in vivo, HCC827 cells which showed relatively high endogenous RCC2 expression were subcutaneously injected into the right ﬂanks (four mice per group). Ten days after tumor inoculation, intratumoral injection of cholesterol-conjugated RCC2 siRNAs (siRNA1 and siRNA2) and their negative controls (10 nmol siRNA in 0.1 ml saline buffer) were used for in vivo siRNA delivery once every 3 days for 3 weeks(25, 26). RCC2 siRNA treatment significantly decreased tumor growth, as shown by significantly reduced tumor size and weight compared with control group (Supplementary Fig. 4A-C). To confirm that RCC2 was knocked down in vivo by siRNA, we measured RCC2 expression by IHC staining and western blotting and found that it was significantly decreased in RCC2 siRNA treated xenografts (Supplementary Fig. 4D-E).

To evaluate whether RCC2 could promote tumor metastasis in vivo, we injected A549-Vec and A549-RCC2 in the tail vein intravenously of nude mice (four mice per group), respectively.

Eight weeks after injection, mice were sacrificed and the metastatic nodules formed on the lung surfaces were examined. As showed in Fig. 5G, the mice injected with A549-Vec cells formed fewer nodules on the lung surfaces than the mice injected with A549-RCC2 cells (P<0.05, independent Student’s t test). Metastatic nodules on the surfaces of mice lungs were confirmed by H&E staining while the expression level of RCC2 in the nodules was also verified by the IHC staining (Fig. 5H). Therefore, our data demonstrated that high RCC2 expression enhanced tumor growth and metastasis in vivo, which was consistent with our in vitro and clinical findings.

RCC2 activated JNK pathway to induce EMT and extracellular matrix remodeling of LUAD cells

JNK pathway is critical for the progression and maintenance of phenotypic and cellular changes associated with EMT(27). In order to further explore the molecular mechanisms of RCC2, we evaluated the possibility of RCC2 contributing to EMT and extracellular matrix remodeling by regulating JNK signaling. We observed that increased expression of activation JNK1/2 in RCC2- transfected cells by western blot analysis (Fig. 6A). As expected, opposite expression pattern of JNK1/2 was examined in RCC2-silenced cells (Fig. 6B). Nevertheless, there was no obvious influence of RCC2 on ERK and p38 phosphorylation. In addition, a significant positive correlation between the overexpression of RCC2 and p-JNK1/2 was evaluated in the same cohort of TMAs containing 120 human LUAD tissues (119 specimens available) (Supplementary Fig. 5). The LUAD samples had high RCC2 expression, which correlated with high JNK1/2 activation (representative case 2). Accordingly, LUAD samples with low RCC2 expression showed reduced JNK1/2 activation (representative case 1) (Supplementary Table 3). These results suggested the involvement of JNK pathways in the effects of RCC2 on LUAD. To confirm that the aggressive effect of RCC2 was mediated by the activation of the JNK1/2, the JNK inhibitor SP600125 was used in A549-RCC2 cells. The results showed that SP600125 could effectively decrease expression levels of phosphorylated JNK1/2 induced by RCC2 in A549-RCC2 cells (Fig. 6C). Wound-healing, migration assays and invasion Transwell assay demonstrated that migratory and invasive ability of A549-RCC2 cells were dramatically inhibited after SP600125 treatment (Fig. 6D-E). In addition, SP600125 also inhibited the RCC2-induced EMT and the expression of MMP-2 and MMP-9 (Fig. 6F). Immunofluorescent staining also confirmed the changes of EMT markers (E-cadherin and N-cadherin) after SP600125 treatment in the A549-RCC2 cells (Fig. 6G). These data, taken together, provided evidence that MAPK-JNK signaling was responsible for the RCC2-induced metastasis in LUAD cells (Fig. 6H).

Discussion

LUAD is considered to be a multi-step process, involving a premalignant lesion to adenocarcinoma in situ, followed by invasive adenocarcinoma(28). Notably, the average 5 year survival rate for LUAD is poor and tumor metastasis is the major cause of LUAD-related death. A better understanding of the underlying molecular mechanisms of LUAD development and metastasis is highly desirable for improvement of clinical management. The gene RCC2, located on chromosome 1p36, has been reported to play a potential role in tumorigenesis. To our knowledge, the abnormalities of RCC2 was associated with gastric carcinoma(18), colorectal cancer(22) directly, and basal cell carcinoma (BCC)(20), melanoma(19), non-small cell lung cancer (NSCLC)(21) indirectly. Recent study demonstrated that RCC2 formed a complex with the

regulator of actin filament assembly cortactin(29), which had been linked to invasive cancers including colorectal cancer, melanoma and glioblastoma, making cortactin as an invasive cancers biomarker(30). RCC2 also acted as a dual activity-limiter of Rac1 and Arf6 to regulate adhesion complex formation, cell spreading and directional migration(14). Although there was no direct evidence of RCC2 involving in tumor metastasis, all these RCC2 interacting proteins, including cortactin, Rac1 and Arf6, had been reported to have direct association with tumor invasiveness and metastasis already(31-34). It was noteworthy that these findings gave the potential links between RCC2 and cancer metastasis. In this study, we first demonstrated that RCC2 was frequently overexpressed in LUAD and RCC2 overexpression was an independent prognostic factor for poor survival of patients with LUAD.

In the present study, western blotting results showed that the majority of the LUAD tissues examined had high levels of RCC2 expression. Subsequently, IHC staining on TMAs which contained 122 pairs of LUAD specimens and matched nontumor tissues was performed to validate the frequent overexpression of RCC2 in 88/122 (72.13%) of LUADs, which was significantly correlated with lymph node metastasis and a more advanced clinical stage, suggesting that upregulated expression of RCC2 in LUAD might facilitate the metastatic phenotype. Further, the analysis of a larger cohort of TCGA RNA-seq expression data that contained 488 lung adenocarcinoma and 58 normal lung tissues confirmed that RCC2 was frequently overexpressed in LUAD tissues versus normal tissues. Moreover, survival analysis showed its important role in the cancer development that patients with RCC2 overexpression had poorer overall survival using multiple datasets. Furthermore, univariate and multivariate Cox regression analysis demonstrated that RCC2 was an independent prognostic factor for poorer outcome in LUAD patients using TCGA expression dataset, suggesting that RCC2 expression could be used as a potential predictor as it is intimately involved in LUAD progression. These findings underscore a potentially important role of RCC2 as an underlying biological mechanism in the development and progression of LUAD.

To clarify the biological function of RCC2 in regulating LUAD cell motility and invasiveness, a series of in vitro and in vivo assays were conducted. The results showed that, the ectopic overexpression of RCC2 could substantially promote cell proliferation, migration and invasiveness. On the contrary, gene silencing of RCC2 by siRNA inhibited its effect on cell growth, motility and invasion. In subcutaneous injection and the tail vein injection mouse models, overexpression of RCC2 resulted in a significant increase in the tumor growth and in the number of lesions of lung metastasis, while knockdown of RCC2 by intra-tumor injection of cholesterol-conjugated RCC2 siRNA significantly inhibited tumorigenicity in vivo. All these findings strongly supported that overexpression of RCC2 played an important role in LUAD invasion and metastasis.

Metastasis represented a multi-step cell-biological process termed the invasion-metastasis cascade including two key events-EMT and extracellular matrix remodeling (35). EMT, involved in normal embryonic morphogenesis initially, played a pivotal role in tumor invasion and metastasis during tumor progression by suppressing expression of epithelial markers and inducing expression of mesenchymal markers, as well as transcription factors(36). In our studies, we found that overexpression of RCC2 had a significant impact on EMT in vitro and in vivo and upregulated the expression of EMT transcription factors Snail and Slug. In contrast, opposite results were

found in RCC2 silencing LUAD cells in vitro. Clinical relevance analysis indicated that there were significant correlations between the expression of RCC2 and EMT markers in LUAD samples. In addition, recent studies showed that RCC2-depleted colon cancer cells appeared more distinctly epithelial-like(22) which was consistent with our results that RCC2 could induce EMT. Besides, matrix metalloproteinases (MMPs), as modulators of the tumor microenvironment, were essential for extracellular matrix remodeling associated with tumorigenesis and tumor metastasis(37, 38). As expected, our studies showed that overexpression of RCC2 could stimulate the expression of MMP-2 and MMP-9. More importantly, using the LUAD samples of TCGA dataset, the results that RCC2 expression was significantly associated with the expression of N-cadherin, Snail, MMP-2 and MMP-9 confirmed our experimental data again. Thus, LUAD cells overexpressing RCC2 probably undergo EMT and extracellular matrix remodeling to achieve higher motility and invasiveness.

To date, however, the molecular mechanisms by which RCC2 regulated EMT and expression of MMP-2 and MMP-9 to promote cancer cell migration and invasiveness have not been elucidated. Previous studies found that the activation of MAPK-JNK pathway was closely associated with lung tumorigenesis(39, 40). JNK not only could regulate critical EMT genes through inducing key transcription factors to influence the progression of phenotypic changes but also regulated MMPs (such as MMP-1, MMP-2 and MMP-9) which played important roles in cell outgrowth, tissue destruction and inflammation (27, 41-43). However, the detail regulatory mechanisms remain unclear. Our results indicated that the expression of phosphorylated JNK increased or decreased according to overexpression or silence of RCC2. Importantly, RCC2 overexpression was positively correlated with JNK activation in human LUAD samples. Moreover, the enhancement of RCC2 on LUAD cell motility and invasiveness and the RCC2-induced EMT and expression of MMP-2 and MMP-9 were dramatically prevented when JNK was inhibited. Therefore, the role of RCC2 in promoting EMT and extracellular matrix remodeling might be carried out through the activation of the MAPK-JNK signaling pathway, ultimately driving LUAD cells motility and invasiveness. Further work is needed to clarify the mechanisms of how RCC2 activates JNK in detail.

In conclusion, we first demonstrated that RCC2 played an important role in the development and progression of LUAD. Overexpression of RCC2 in LUAD was significantly associated with tumor metastasis and was an independent prognostic factor for poorer outcomes in LUAD patients. Furthermore, functional and mechanistic studies revealed that RCC2 regulated EMT and extracellular matrix remodeling to promote cancer cell motility and tumor metastasis through the activation of the MAPK-JNK signaling. Therefore, our studies suggested that RCC2 may serve as a clinically useful diagnostic and prognostic biomarker, and a potentially therapeutic target in LUAD.

Authors' Contributions

Conception and design: Y. Jin, S. Fu Development of methodology: B. Pang, Y. Jin, S. Fu Acquisition of data: B. Pang, N. Wu, R. Guan, L. Pang, X. Li, L. Tang, S. Li, J. Dai Analysis and interpretation of data: B. Pang, N. Wu, L. Pang, D. Sun, H. Sun, Y. Jin, S. Fu Writing, review, and/or revision of the manuscript: B. Pang, N. Wu, L. Pang, Y. Jin, S. Fu Administrative, technical, or material support: R. Guan, X. Li, L. Tang, Y. Guo, J. Chen, D. Sun, H. Sun, J. Dai, J. Bai, G. Ji, P. Liu, A. Liu, Q. Wang, S. Xiao Study supervision: Y. Jin, S. Fu

References

1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA: a cancer journal for clinicians. 2014;64:9-29. 2. Cancer Genome Atlas Research N. Comprehensive molecular profiling of lung adenocarcinoma. Nature. 2014;511:543-50. 3. Shiraishi K, Kunitoh H, Daigo Y, Takahashi A, Goto K, Sakamoto H, et al. A genome-wide association study identifies two new susceptibility loci for lung adenocarcinoma in the Japanese population. Nature genetics. 2012;44:900-3. 4. Imielinski M, Berger AH, Hammerman PS, Hernandez B, Pugh TJ, Hodis E, et al. Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell. 2012;150:1107-20. 5. Kasinski AL, Slack FJ. miRNA-34 prevents cancer initiation and progression in a therapeutically resistant K-ras and p53-induced mouse model of lung adenocarcinoma. Cancer research. 2012;72:5576-87. 6. Li CM, Chen G, Dayton TL, Kim-Kiselak C, Hoersch S, Whittaker CA, et al. Differential Tks5 isoform expression contributes to metastatic invasion of lung adenocarcinoma. Genes & development. 2013;27:1557-67. 7. Cheung WK, Zhao M, Liu Z, Stevens LE, Cao PD, Fang JE, et al. Control of alveolar differentiation by the lineage transcription factors GATA6 and HOPX inhibits lung adenocarcinoma metastasis. Cancer cell. 2013;23:725-38. 8. Andreassen PR, Palmer DK, Wener MH, Margolis RL. Telophase disc: a new mammalian mitotic organelle that bisects telophase cells with a possible function in cytokinesis. Journal of cell science. 1991;99 ( Pt 3):523-34. 9. Adams RR, Wheatley SP, Gouldsworthy AM, Kandels-Lewis SE, Carmena M, Smythe C, et al. INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow. Current biology : CB. 2000;10:1075-8. 10. Cooke CA, Heck MM, Earnshaw WC. The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis. The Journal of cell biology. 1987;105:2053-67. 11. Gassmann R, Carvalho A, Henzing AJ, Ruchaud S, Hudson DF, Honda R, et al. Borealin: a novel chromosomal passenger required for stability of the bipolar mitotic spindle. The Journal of cell biology. 2004;166:179-91. 12. Mollinari C, Reynaud C, Martineau-Thuillier S, Monier S, Kieffer S, Garin J, et al. The mammalian passenger protein TD-60 is an RCC1 family member with an essential role in prometaphase to metaphase progression. Developmental cell. 2003;5:295-307. 13. Papini D, Langemeyer L, Abad MA, Kerr A, Samejima I, Eyers PA, et al. TD-60 links RalA GTPase function to the CPC in mitosis. Nature communications. 2015;6:7678. 14. Humphries JD, Byron A, Bass MD, Craig SE, Pinney JW, Knight D, et al. Proteomic analysis of integrin-associated complexes identifies RCC2 as a dual regulator of Rac1 and Arf6. Science signaling. 2009;2:ra51. 15. Williamson RC, Cowell CA, Hammond CL, Bergen DJ, Roper JA, Feng Y, et al. Coronin-1C and RCC2 guide mesenchymal migration by trafficking Rac1 and controlling GEF exposure. Journal of cell science. 2014;127:4292-307.

16. Danen EH. Integrin proteomes reveal a new guide for cell motility. Science signaling. 2009;2:pe58. 17. Yenjerla M, Panopoulos A, Reynaud C, Fotedar R, Margolis RL. TD-60 is required for interphase cell cycle progression. Cell cycle. 2013;12:837-41. 18. Matsuo M, Nakada C, Tsukamoto Y, Noguchi T, Uchida T, Hijiya N, et al. MiR-29c is downregulated in gastric carcinomas and regulates cell proliferation by targeting RCC2. Molecular cancer. 2013;12:15. 19. Rendleman J, Shang S, Dominianni C, Shields JF, Scanlon P, Adaniel C, et al. Melanoma risk loci as determinants of melanoma recurrence and survival. Journal of translational medicine. 2013;11:279. 20. Stacey SN, Gudbjartsson DF, Sulem P, Bergthorsson JT, Kumar R, Thorleifsson G, et al. Common variants on 1p36 and 1q42 are associated with cutaneous basal cell carcinoma but not with melanoma or pigmentation traits. Nature genetics. 2008;40:1313-8. 21. Feng N, Ching T, Wang Y, Liu B, Lin H, Shi O, et al. Analysis of Microarray Data on Gene Expression and Methylation to Identify Long Non-coding RNAs in Non-small Cell Lung Cancer. Scientific reports. 2016;6:37233. 22. Bruun J, Kolberg M, Ahlquist TC, Royrvik EC, Nome T, Leithe E, et al. Regulator of Chromosome Condensation 2 Identifies High-Risk Patients within Both Major Phenotypes of Colorectal Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research. 2015;21:3759-70. 23. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC bioinformatics. 2011;12:323. 24. Uhlen M, Bandrowski A, Carr S, Edwards A, Ellenberg J, Lundberg E, et al. A proposal for validation of antibodies. Nature methods. 2016;13:823-7. 25. Hou J, Lin L, Zhou W, Wang Z, Ding G, Dong Q, et al. Identification of miRNomes in human liver and hepatocellular carcinoma reveals miR-199a/b-3p as therapeutic target for hepatocellular carcinoma. Cancer cell. 2011;19:232-43. 26. Hou J, Zhou Y, Zheng Y, Fan J, Zhou W, Ng IO, et al. Hepatic RIG-I predicts survival and interferon-alpha therapeutic response in hepatocellular carcinoma. Cancer cell. 2014;25:49-63. 27. Sahu SK, Garding A, Tiwari N, Thakurela S, Toedling J, Gebhard S, et al. JNK-dependent gene regulatory circuitry governs mesenchymal fate. The EMBO journal. 2015;34:2162-81. 28. Yatabe Y, Matsuo K, Mitsudomi T. Heterogeneous distribution of EGFR mutations is extremely rare in lung adenocarcinoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2011;29:2972-7. 29. Grigera PR, Ma L, Borgman CA, Pinto AF, Sherman NE, Parsons JT, et al. Mass spectrometric analysis identifies a cortactin-RCC2/TD60 interaction in mitotic cells. Journal of proteomics. 2012;75:2153-9. 30. MacGrath SM, Koleske AJ. Cortactin in cell migration and cancer at a glance. Journal of cell science. 2012;125:1621-6. 31. Yang WH, Lan HY, Huang CH, Tai SK, Tzeng CH, Kao SY, et al. RAC1 activation mediates Twist1-induced cancer cell migration. Nature cell biology. 2012;14:366-74. 32. Schmitz AA, Govek EE, Bottner B, Van Aelst L. Rho GTPases: signaling, migration, and invasion. Experimental cell research. 2000;261:1-12. 33. Valderrama F, Ridley AJ. Getting invasive with GEP100 and Arf6. Nature cell biology.

2008;10:16-8. 34. Kashatus DF. Ral GTPases in tumorigenesis: emerging from the shadows. Experimental cell research. 2013;319:2337-42. 35. Valastyan S, Weinberg RA. Tumor metastasis: molecular insights and evolving paradigms. Cell. 2011;147:275-92. 36. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139:871-90. 37. Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell. 2010;141:52-67. 38. Oh J, Takahashi R, Kondo S, Mizoguchi A, Adachi E, Sasahara RM, et al. The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis. Cell. 2001;107:789-800. 39. Wang IC, Ustiyan V, Zhang Y, Cai Y, Kalin TV, Kalinichenko VV. Foxm1 transcription factor is required for the initiation of lung tumorigenesis by oncogenic Kras(G12D.). Oncogene. 2014;33:5391-6. 40. Shih MC, Chen JY, Wu YC, Jan YH, Yang BM, Lu PJ, et al. TOPK/PBK promotes cell migration via modulation of the PI3K/PTEN/AKT pathway and is associated with poor prognosis in lung cancer. Oncogene. 2012;31:2389-400. 41. Park CH, Lee MJ, Ahn J, Kim S, Kim HH, Kim KH, et al. Heat shock-induced matrix metalloproteinase (MMP)-1 and MMP-3 are mediated through ERK and JNK activation and via an autocrine interleukin-6 loop. The Journal of investigative dermatology. 2004;123:1012-9. 42. Cheng CY, Hsieh HL, Hsiao LD, Yang CM. PI3-K/Akt/JNK/NF-kappaB is essential for MMP-9 expression and outgrowth in human limbal epithelial cells on intact amniotic membrane. Stem cell research. 2012;9:9-23. 43. Lee YS, Lan Tran HT, Van Ta Q. Regulation of expression of matrix metalloproteinase-9 by JNK in Raw 264.7 cells: presence of inhibitory factor(s) suppressing MMP-9 induction in serum and conditioned media. Experimental & molecular medicine. 2009;41:259-68.

Table 1. Correlation between RCC2 expression and clinicopathological features in LUAD RCC2 All cases Low expression High expression P valuea Gender 0.301 Female 59 19 (32.2%) 40 (67.8%) Male 63 15 (23.8%) 48 (76.2%) Ageb 0.603 ≥60.8c 61 16 (26.2%) 45 (73.8%) <60.8 59 18 (30.5%) 41 (69.5%) pT status 0.002 1 32 16 (50%) 16 (50%) 2 67 17 (25.4%) 50 (74.6%) 3 18 1 (5.6%) 17 (94.4%) 4 5 0 (0%) 5 (100%) pN statusb 0.004 0 58 22 (37.9%) 36 (62.1%) 1 18 1 (5.6%) 17 (94.4%) 2 15 1 (6.7%) 14 (93.3%) 3 5 0 (0%) 5 (100%) Stage 0.001 I 17 11 (64.7%) 6 (35.3%) II 79 18 (22.8%) 61 (77.2%) III 26 5 (19.2%) 21 (80.8%) aThe Pearson chi-squared test or Fisher's exact test was used for statistical analysis. bPartial data not available; statistics based on available data. cThe mean age at diagnosis is 60.8 years in LUAD patients. Samples are divided into two groups based on the mean age. RCC2, regulator of chromosome condensation 2; LUAD, lung adenocarcinoma.

Table 2. Univariate and multivariate Cox regression analysis of different prognosis factors in patients with LUAD from TCGA database Univariate analysisa Multivariate analysisa Factors Sample HR 95%CI P valueb HR 95%CI P valueb Total 294 RCC2 expression 0.012 0.017 Low expression 184 High expression 110 1.7 1.1-2.6 1.7 1.1-2.6 Age 0.457 0.189 ≤65c 143 >65 151 1.2 0.8-1.8 1.4 0.9-2.2 Gender 0.751 0.566 Female 147 Male 147 0.9 0.6-1.4 0.9 0.6-1.4 pT status T1 83 T2 170 1.4 0.8-2.5 0.299 1.2 0.6-2.3 0.554 T3 25 2.6 1.1-6.2 0.028 2.0 0.8-5.3 0.151 T4 16 3.5 1.5-8.0 0.003 1.5 0.5-4.3 0.448 pN status N0 181 N1 63 1.8 1.1-3.0 0.015 1.7 0.8-4.0 0.2 N2+N3 50 2.4 1.4-4.1 <0.001 0.8 0.2-2.4 0.638 pM status M0 275 M1 19 1.5 0.7-3.1 0.305 NAd Stage I 147 II 72 1.7 1.0-2.9 0.073 0.9 0.4-2.4 0.91 III 56 3.2 1.9-5.4 <0.001 3.4 1.0-11.8 0.058 IV 19 2.3 1.0-5.1 0.04 2.0 0.7-5.8 0.186 aCox regression model. bLog-rank test. cThe mean age at diagnosis is 65 years in LUAD patients from TCGA database. Samples are divided into two groups based on the mean age. dSince M1 completely overlapped with Stage IV, the P value failed to calculate in Cox regression model. LUAD, lung adenocarcinoma; HR, hazard ratio; CI, confidence interval.

Figure Legends:

Figure 1. Expression of RCC2 in LUAD and its prognostic significance in LUAD patients. (A) Representative images of RCC2 IHC staining in LUAD tissue and (B) adjacent normal tissue (magnification ×400). Scale bars=200 μm. (C) RCC2 expression was compared between 488 LUAD tissues and 58 normal lung tissues. (D) The expression of RCC2 protein was detected by western blotting in six pairs of LUAD tissues (T) and their adjacent nontumor tissues (N). (E) Kaplan-Meier survival analysis according to RCC2 expression in 122 LUAD patients from TMAs (log-rank test). Probability of survival of patients: low expression of RCC2, n=34; overexpression of RCC2, n=88 (P=0.003). (F) Kaplan-Meier survival analysis according to RCC2 expression in 420 patients with LUAD (log-rank test) from TCGA database. Probability of survival of patients: low expression of RCC2, n=262; overexpression of RCC2, n=158 (P=0.032).

Figure 2. RCC2 promoted cell proliferation, migration and invasion in vitro. (A) Endogenous RCC2 expression status in five LUAD cell lines. (B) Western blot analysis showing ectopic expression of exogenous RCC2 in RCC2-transfected A549 and NCI-H1650 cells. (C) Effect of RCC2 overexpression on A549 and NCI-H1650 cell growth. Results were expressed as mean ± SD of three independent experiments (**P<0.01; ***P<0.001, independent Student’s t test). (D) Wound-healing assay indicating RCC2 overexpression enhanced A549 cell migration (magnification ×40). Scale bars=500 μm. (E-H) Transwell migration and invasion assay showing that RCC2 overexpression promoted cell migration (E-F) and invasion (G-H) in A549 and NCI-H1650 cells (magnification ×100). Scale bars=500 μm. Results were summarized as mean ± SD of three independent experiments (***P<0.001, independent Student’s t test).

Figure 3. Silencing of RCC2 inhibited cell proliferation, migration and invasion in vitro. (A) RCC2 expression was efficiently knocked down by two targeted siRNAs (siRNA1 and siRNA2) in A549-RCC2 cells with stable RCC2 expression and HCC827 cells as detected by western blotting. Silencing RCC2 expression suppressed cell growth (B), and cell migration determined by the wound-healing assay (C-D) (magnification ×40). Scale bars=500 μm. Suppression of RCC2 reduced cell migration and invasion capacity of A549-RCC2 (E) and HCC827 (F) cells in the Transwell migration and invasion assay (magnification ×100). Scale bars=500 μm. Results were expressed as mean ± SD of three independent experiments (**P<0.01; ***P<0.001, independent Student’s t test).

Figure 4. RCC2 induced EMT and the expression of MMP-2 and MMP-9 in LUAD cells. (A) Western blot analysis showed decreased expression of epithelial makers (β-catenin and E-cadherin) and increased expression of mesenchymal markers (N-cadherin and α-SMA), EMT-related transcription factors (Snail and Slug), and two matrix metalloproteinases (MMP-2 and MMP-9) in RCC2-transfected A549 and NCI-H1650 cells compared with that in control cells. (B) Knockdown of RCC2 in A549-RCC2 and HCC827 cells resulted in upregulation of epithelial markers (β-catenin and E-cadherin) and downregulation of mesenchymal markers (N-cadherin and α-SMA), EMT-related transcription factors (Snail and Slug), and two matrix metalloproteinases (MMP-2 and MMP-9) by western blotting. (C) Immunofluorescent staining assay showed a downregulated expression of β-catenin and an upregulated expression of N-cadherin in A549-RCC2 cells (magnification ×400). Scale bars=100 μm. (D) The correlation analysis was performed between RCC2 expression with the expression of N-cadherin, Snail, MMP-2 and

MMP-9 in LUAD samples from TCGA dataset (P<0.001).

Figure 5. RCC2 promoted xenograft tumor growth and metastatic potential in mouse models. (A) Tumor volumes were compared between xenograft tumors (n=5) induced with A549-RCC2 and A549-Vec cells (*P<0.05; ***P<0.001). (B) Images of the xenograft tumors subcutaneously injected with A549-Vec and A549-RCC2 cells. (C) Tumor weights were compared between A549-Vec and A549-RCC2 groups. (D) Representative H&E staining images (left panel) (magnification ×100, scale bars=500 μm) and IHC images of RCC2 staining (right panel) (magnification ×400, scale bars=200 μm) in section of xenograft tumors were showed. (E) qRT-PCR analysis showing RCC2 expression levels in xenograft tumors from A549-Vec and A549-RCC2 groups (F) Expression of EMT markers (E-cadherin and N-cadherin) was detected by western blotting using xenograft tumors from A549-Vec (n=4) and A549-RCC2 (n=5) groups. (G) Metastatic nodules on the lung surfaces of mice were summarized (*P<0.05, independent Student’s t test). (H) Representative images of metastatic nodules on the surface of lung in nude mice (left panel). H&E staining (median panel) (magnification ×100, scale bars=500 μm) and IHC staining with RCC2 (right panel) (magnification ×400, scale bars=200 μm) were performed on section of lung metastatic nodules.

Figure 6. RCC2 induced EMT and the expression of MMP-2 and MMP-9 to achieve LUAD cell aggressiveness via MAPK-JNK signaling. (A-B) The expression of p-JNK, JNK, p-ERK, ERK, p-p38, p38 was detected by western blot analysis in RCC2 overexpression A549 cells (A) and RCC2 silencing A549-RCC2 cells (B). GAPDH was used as the loading control. (C) Western blot analysis showed that the JNK inhibitor SP600125 effectively decreased expressions of p-JNK induced by RCC2 in A549-RCC2 cells. (D) Would-healing assay showed that the enhanced cell migration capacity in A549-RCC2 cells was suppressed by SP600125 (magnification ×40). Scale bars=500 μm. (E) Transwell migration and invasion assay showing that SP600125 could inhibit RCC2-induced cell migration and invasion in A549-RCC2 cells (magnification ×100). Scale bars=500 μm. Results were summarized as mean ± SD of three independent experiments (***P<0.001, independent Student’s t test). (F) Western blotting showed that after treatment of SP600125 in A549-RCC2 cells, the expression of the E-cadherin increased and the expressions of mesenchymal markers (N-cadherin and α-SMA), EMT-related transcription factors (Snail), and two matrix metalloproteinases (MMP-2 and MMP-9) decreased. (G) Immunofluorescent staining showed an upregulated expression of E-cadherin and a downregulated expression of N-cadherin in A549-RCC2 cells after treatment by JNK inhibitor SP600125 (magnification ×400). Scale bars=100 μm. (H) Schematic representation of RCC2 induced EMT and the expression of MMP-2 and MMP-9 via MAPK-JNK pathway in LUAD metastasis.

Overexpression of RCC2 Enhances Cell Motility and Promotes Tumor Metastasis in Lung Adenocarcinoma by Inducing Epithelial-Mesenchymal Transition

Bo Pang, Nan Wu, Rongwei Guan, et al.

Clin Cancer Res Published OnlineFirst June 12, 2017.

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