NF45 promotes esophageal squamous carcinoma cell invasion by increasing Rac1 activity through 14-3-3ε protein

NF45 promotes esophageal squamous carcinoma cell invasion by increasing Rac1 activity through 14-3-3ε protein

Accepted Manuscript NF45 promotes esophageal squamous carcinoma cell invasion by increasing Rac1 activity through 14-3-3ε protein Yao Wen-Jian, Tong s...

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Accepted Manuscript NF45 promotes esophageal squamous carcinoma cell invasion by increasing Rac1 activity through 14-3-3ε protein Yao Wen-Jian, Tong song, Tan Jun, Xu Kai-Ying, Wang Jian-Jun, Wang Si-Hua PII:

S0003-9861(18)30582-4

DOI:

https://doi.org/10.1016/j.abb.2018.12.012

Reference:

YABBI 7887

To appear in:

Archives of Biochemistry and Biophysics

Received Date: 25 July 2018 Revised Date:

4 December 2018

Accepted Date: 10 December 2018

Please cite this article as: Y. Wen-Jian, T. song, T. Jun, X. Kai-Ying, W. Jian-Jun, W. Si-Hua, NF45 promotes esophageal squamous carcinoma cell invasion by increasing Rac1 activity through 14-3-3ε protein, Archives of Biochemistry and Biophysics (2019), doi: https://doi.org/10.1016/j.abb.2018.12.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT NF45 promotes esophageal squamous carcinoma cell invasion by increasing Rac1 activity through 14-3-3ε protein. Yao Wen-Jian; Tong song; Tan Jun; Xu Kai-Ying; Wang Jian-Jun*; Wang Si-Hua* Department of Thoracic Surgery, Union Hospital, Tongji Medical College, Huazhong

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University of Science and Technology, Wuhan, China

Correspondence to: Dr. Wang Jian-Jun, and Dr. Wang Si-Hua. Department of

Science and Technology, Wuhan 430022, China;

Tong song: [email protected] Tan Jun: [email protected] Xu Kai-Ying: [email protected]

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E-mail: Yao Wen-Jian: [email protected]

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Thoracic Surgery, Union Hospital, Tongji Medical College, Huazhong University of

Wang Jian-Jun: [email protected]

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Wang Si-Hua: [email protected]

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Tel: :Wang Si-Hua: +86-13517232043 and Wang Jian-Jun: +86-15827327278

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Fox: Wang Si-Hua: +86-02785351615 and Wang Jian-Jun: +86-02785871828

Running title: NF45 promotes esophageal squamous carcinoma cell invasion through 14-3-3ε protein

ACCEPTED MANUSCRIPT Abstract Nuclear factor 45 (NF-45) has been found to be markedly upregulated in several cancers, including esophageal squamous cell carcinoma (ESCC). However, the molecular mechanisms underlying its functions remain unclear. In this study, we

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confirm that overexpression of NF45 was frequently detected in ESCC tissues and was associated with poor outcome. Overexpression studies revealed that NF-45 promoted cell growth and invasion and upregulated Rac1/Tiam1 signaling via 14-3-3ε protein in ESCC cell lines. This novel mechanism linking upregulated NF45

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expression to increased 14-3-3ε/Rac1/Tiam1 signaling and subsequent growth and

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invasion in ESCC may aid in identification of new therapeutic targets for this disease.

Keywords: NF45; 14-3-3ε; Esophageal squamous cell carcinoma (ESCC); Invasion;

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Prognosis

ACCEPTED MANUSCRIPT Introduction Esophageal squamous cell carcinoma (ESCC) is ranked as the fourth leading cause of cancer death in China [1]. Despite recent advances in treatment, the prognosis for patients with ESCC remains poor. Therefore, a better understanding of molecular

potential therapeutic targets for ESCC.

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mechanisms underlying the development of ESCC is essential for identifying

Nuclear factor 45 (NF-45), also known as interleukin enhancer-binding factor 2

(ILF2), is a subunit of NF-AT (nuclear factor of activated T cells) that belongs to the

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double-stranded RNA-binding protein family [2]. NF45 is a regulatory subunit of

complexes with NF90/110 involved in RNA transcription and translation [3, 4]. NF45

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was initially identified as together with its binding partner NF90/NF110 to regulate IL-2 transcription [5]. NF45 is ubiquitously expressed in various cell types and regulates gene expression at multiple levels including RNA transport, transcription, and translation [6]. Multiple studies have shown that NF45 is frequently overexpressed in several types of human cancer, including lung cancer [7], cervical

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cancer [8], pancreatic adenocarcinoma [9], hepatocellular carcinoma [10], multiple myeloma [11], and esophageal cancer [12]. The expression of NF45 is increased in ESCC and high NF45 expression was suggested to be a prognostic factor for ESCC

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patients' poor survival [12]. However, the precise molecular mechanisms of NF45 in ESCC development remains virtually unknown. We hypothesized that NF45 promotes ESCC development and progression by increasing Tiam1/Rac1 activity through

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14-3-3ε protein.

14-3-3 family comprises seven conserved isoforms (β, ε, γ, η, τ, ζ, σ). As a member of the 14-3-3 protein family, 14-3-3ε serves functions in the regulation of the apoptosis and cell cycle[13, 14]. 14-3-3ε is significantly associated with poor survival rates in colorectal cancer and hepatocellular carcinoma patients [15, 16]. Rac1 is a member of the Rho family of small GTPases that regulates many diverse cellular functions including cell proliferation, migration, and cytoskeletal reorganization. Rac1 activity is regulated by the GTPase activating proteins (GAPs), which exchange GTP to GDP and inactivate Rac1, and guanine nucleotide exchange

ACCEPTED MANUSCRIPT factors (GEFs), which stimulate the conversion of bound GDP for GTP and activate Rac1 [17, 18]. Tiam1 is a guanine nucleotide exchange factors (GEFs) that activates the Rho-family GTPase Rac1 [19, 20]. Increasing evidence suggests that Rac1 is activated in various cancers to promote proliferation, invasion, and metastasis [21].

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Previous studies have shown that 14-3-3 protein overexpression results in the activation of Tiam1 and Rac1 and mediates cell migration [22]. Although activation of Tiam1/Rac1 was observed in certain cancers, the upstream signaling involved in

Tiam1/Rac1 activation is largely unknown. Thus, we hypothesized that Tiam1/Rac1

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may also be involved in NF45 mediated ESCC development and progression. In the present study, we report that NF45 overexpression is associated with ESCC

Materials and methods

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malignancy, and Rac1 is activated by 14-3-3ε protein.

ESCC tissue specimens and cell lines. The ESCC tumors and matched adjacent normal tissues were collected from 75 ESCC patients who underwent surgery as their

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first treatment between 2016 and 2017 at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). Tissue specimens were collected from patients who signed informed consent. The samples used in this

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study were approved by the Human Research Committee of Huazhong University of Science and Technology. The human ESCC lines EC18, Eca109, KYSE150, KYSE180 and normal esophageal epithelial cell line Het-1A were purchased from the

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Cell Bank of Type Culture Collection of Chinese Academy of Sciences and maintained in RPMI-1640 medium (Hyclone, USA) supplemented with 10% fetal calf serum (Gibco, USA).

Antibodies and materials. Antibody against NF45 (ab28772), 14-3-3ε (ab137862), Tiam1 (ab211518) and Rac1 (ab155938) were purchased from Abcam (Abcam, USA); antibodies against β-actin (4970) and GAPDH (5174) were from Cell Signaling Technology (USA).

ACCEPTED MANUSCRIPT Plasmid constructs and transfection. Full-length human NF45 cDNA was amplified, cloned into pcDNA3.1(+) expression vector (Invitrogen, USA), and then transfected into Eca109 and KYSE180 cells using Lipofectamine 2000 (Invitrogen, USA). Cells transfected with empty vector were used as negative controls. Lentiviruses containing

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shRNAs targeting NF45 and 14-3-3ε were purchased from GeneChem (GeneChem, China). The shNF45 target sequence was 5'- TCGACAGGTGGGATCCTATAA -3', sh14-3-3ε target sequence was 5'- GCTTAGGTCTTGCTCTCAATT -3'. Cells

transfected with scrambled shRNA (GeneChem, China) were used as negative

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controls.

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Quantitative real-time PCR. Total RNA was extracted and reverse-transcribed from clinical samples using TRIzon Reagent and SuperRT cDNA Synthesis Kit (CWBIO, China). Real-time qPCR was performed using SYBR® Premix Ex Taq™ Kit (Takara, Japan). 18S rRNA was used as an internal control. The primer sequences are listed in the Supporting Methods. The relative mRNA expression level was normalized as

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previously described [23][20]. The relative expression level of target gene (2−∆∆Ct) was normalized to the endogenous reference (∆Ct). NF45 levels in tissues that were equal and/or lower than the median value (50th) were defined as ‘low’, and levels

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higher than the median value were defined as ‘high’.

Western blotting. Western blot assay was performed as previously described [24, 25],

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using anti-NF45 and anti-14-3-3ε antibodies.

TCGA data analyses. For TCGA data, the gene expression data of 90 esophageal squamous cell carcinoma patients were downloaded from the TCGA Web site. NF45 mRNA levels from ESCC patients (n=90) were sorted in descending order and divided into a high-expressing group (n=48) and a low-expressing group (n=42) to identify survival differences between the two groups.

Colony formation assay and MTT assay. For colony formation assays, 500 cells

ACCEPTED MANUSCRIPT were plated in 35-mm dishes. About 14 days later, cells were fixed and then stained by crystal violet. The colonies with a diameter ≥ 2mm were counted. For the MTT assay, cells were plated at an density of 4 × 103/ well in 96-well plates. After treatment with MTT and DMSO (Sigma, USA), the absorbance of each well was measured in a

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microtiter reader at 490 nm.

Xenografted tumor model. Experimental procedures were approved by the

Institutional Animal Care and Use Committee of Huazhong University of Science and

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Technology. The BALB/c nude mice were randomly divided (5 per group) and

injected subcutaneously with KYSE180 cells (6×106 cells). Mice were euthanized at

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day 21 after injection, and the subcutaneous tumors were weighted.

Boyden chamber invasion assay and wound-healing assay. For cell invasion assays, 5 × 105 cells were seeded on the top chamber with a Matrigel (BD Biosciences, USA) -coated Transwell (Costar, UK). 20% fetal bovine serum (Invitrogen, USA) was

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added to the lower chamber. After a 24-hour incubation, cells on the lower surface of the membrane were stained with crystal violet and counted. For wound-healing assay, cells were seeded into 6-well dishes. After a 12-hour incubation, the scrape wounds

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were made for each sample. Eca109 and KYSE180 cells were counted directly under

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a microscope when the wound was scratched and 24 hours after.

Rac1 activation assay. Rac1 regulates molecular events by cycling between an inactive GDP-bound form and an active GTP-bound form. In its active (GTP-bound) state, Rac1 binds specifically to the p21-binding domain (PBD) of p21-activated protein kinase (PAK) to control downstream signaling cascades. Rac-GTP pulldown assay were performed on Eca109 and KYSE180 cells as previously described [19]. PAK PBD (p21-binding domain of p21-activated protein kinase) agarose beads were used to pull down the active form of Rac1. Assays were analysed using the small GTPase activation assays kit (STA-401-1, Cell Biolabs) following the manufacturer's instructions.

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Tiam1-activity assay. Active Tiam1 was pulled down using Rac1 G15A agarose beads. Assays were performed using the Active Rac-GEF Assay Kit (Tiam1)

Immunohistochemistry and immunofluorescence staining.

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(STA-422, Cell Biolabs) according to the manufacturer’s instructions.

IHC analysis was

performed to study NF45 expression in 77 human paraffin-embedded ESCC samples. The procedure was performed as previously described [19]. The scores were defined

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according to the staining intensity (3 = strong, dark brown staining; 2 = moderate,

dark yellow/light brown staining; 1=weak, light yellow staining; and 0 = negative)

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multiplied by the extent of cells that stained positive (75–100% positive tumor cells = 4, 50–74% = 3, 25–49% = 2, 1–24% = 1, 0% = 0), leading to scores from 12 to 0. For IF staining, KYSE180 cells were fixed and incubated in blocking buffer for 30 min. Primary antibody (rabbit anti-14-3-3ε, 1:200) was detected with an anti-rabbit

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secondary antibody (SA00004-8, Proteintech, China).

Statistical analysis. EmpowerStats statistical software program (http://www.empowerstats.com/) was used for statistical analyses. Statistical tests for

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data analysis included χ2 (clinicopathological features) and Student’s two-tailed t- test (cell biological experiments). Kolmogorov-Smirnov test was used evaluated for the normality of distribution. Survival analyses were assessed by Kaplan–Meier method

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and log-rank tests. Cox proportional hazards regression model was used in multivariate statistical analysis. P < 0.05 was considered statistically significant.

Results

NF45 is upregulated in ESCC and correlates with poor prognosis Previous study have shown that NF45 is upregulat in ESCC tissues [12], so we aimed to confirm these findings and then further study the mechanism of how increased NF45 affects cancer progression We determined the expression of NF45 in ESCC specimens paired with the normal tissues. NF45 protein expression was examined by

ACCEPTED MANUSCRIPT western blotting and immunohistochemical staining. Typical western blotting of NF45 in ESCC tissues and the matched adjacent non-tumor tissues was shown in Figure 1A. By using qRT-PCR, we found that NF45 was overexpressed in >55% of the ESCC tissues compare to adjacent normal tissue (> 2-fold increase in 45 out of 77 cases)

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(Figure 1B). Additionally, as shown in Figures 1C and D, the immunostaining intensity of NF45 in ESCC tissues was frequently higher than in normal tissues.

Moreover, the expression of NF45 was significantly associated with local invasion (P = 0.02), but not with other clinicopathological features (Table 1). Furthermore,

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upregulation of NF45 was associated with decreased disease-free survival (DFS) rates in 77 ESCC cases (Figures 1D). We also examined NF45 mRNA expression in TCGA

rates in TCGA dataset (Figures 1E).

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dataset. Overexpression of NF45 was associated with poorer overall survival (OS)

NF45 promotes ESCC cell proliferation

To assess the potential role of NF45 in ESCC proliferation, we determined the protein

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level of NF45 in ESCC cell lines and found the highest expression of NF45 in ECA109 cells and the lowest expression in KYSE180 cells (Figures 2A). NF45 cDNA or NF45 shRNA were used for upregulating or downregulating NF45 expression.

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After transfection, NF45 cDNA effectively upregulated the expression of NF45 in KYSE180 cells, and NF45 shRNA suppressed the expression of NF45 in ECA109 cells (Figures 2B). Overexpression of NF45 resulted in an increase in cell growth rate

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(Figures 2C), frequency of colony formation (Figure 2D), and the formation of tumors (Figure 2E). Consistent with these results, knockdown of NF45 suppressed cell growth rate and colony formation (Figure 2C – 2D).

NF45 promotes ESCC cell invasion The role of NF45 in tumor cell migration and invasion was investigated. By the wound-healing assay, we found that the migration of cancer cells overexpressing NF45 increased compared to control cells (Figure 3A). This observation was confirmed by the Boyden chamber invasion assay, the overexpression of NF45

ACCEPTED MANUSCRIPT resulted in increased invasion rate compared to control cells (Figure 3B). Consistent with these results, knockdown of NF45 suppressed cellular migration and invasion (Figure 3A – 3B).

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NF45 activates Tiam1-Rac1 signalling Rac1, as a cytoskeleton modulator, is critical for a number of cellular activities,

including migration, proliferation and adhesion [18, 27]. To test whether NF45 might regulate Rac1 activity, we performed PAK binding domain (PAK-PBD) pull down

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assays to monitor changes in Rac1 activation. Cells overexpressing NF45 had a high Rac-GTP levels compared to control cells (Figure 4A). Rac1 is a nucleotide-free

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mutant Rac1(G15A) that has a high affinity for activated GEFs, such as active Tiam1. Therefore, we used Rac1 G15A agarose beads that interacts with active Tiam1 to test the changes in Tiam1 activation. Cells overexpressing NF45 had a dramatic upregulation in the levels of active Tiam1 (Figure 4A). These observations

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demonstrate that NF45 activates Tiam1-Rac1 signalling in ESCC cells.

NF45 upregulates 14-3-3ε expression

14-3-3 proteins (β, ε, γ, η, τ, ζ, σ) act as adaptors that interact with various proteins

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[28]. Because previous studies showed that 14-3-3 protein could bind to Tiam1 and activate Rac1, we hypothesized that NF45 may promote ESCC cell invasion by activating the Tiam1-Rac1 signaling through 14-3-3 proteins. Thus, we assessed

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14-3-3 mRNA expression to see which isoform of 14-3-3 would be regulated by NF45. As shown in Figure 4B, overexpression of NF45 upregulated 14-3-3ε mRNA expression. It was confirmed by western blotting that overexpression of NF45 upregulated 14-3-3ε protein expression (Figure 4C). Immunofluorescent staining confirmed that the increased expression of 14-3-3ε was detected in KYSE180 cells over-expressing NF45 (Figure 4D). Next, we investigate the correlation between NF45 and 14-3-3ε mRNA levels in the cohort of ESCC samples. The results confirmed that 14-3-3ε mRNA expression was significantly correlated with NF45 expression (R = 0.377, P < 0.01, Figure 4E). Analysis of the TCGA esophageal cancer

ACCEPTED MANUSCRIPT data also confirmed this correlation (R = 0.371, P = 0.0003, Figure 4F). 14-3-3ε is involved in NF45-induced Rac1 activation, contributing to the process of ESCC cell invasion To test whether 14-3-3ε is involved in NF45-regulated ESCC cell proliferation and

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invasion, we inhibited 14-3-3ε expression by shRNA (Figure 5A). 14-3-3ε-shRNA decreased the effects of NF45 on promoting cell growth (Figure 5B-D), migration

(Figure 5E), and invasion (Figure 5F). 14-3-3ε-shRNA also decreased the effects of

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NF45 on promoting Tiam1-Rac1 activation in KYSE180 cells (Figure 5G).

Discussion

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In this study, we provide multiple pieces of evidences that NF45 is a critical factor in promoting the tumorigenesis of ESCC. We found that overexpression of NF45 was significantly associated with local invasion, poor DFS rate and poor overall survival rate. Functional studies showed that overexpression of NF45 promoted cell growth

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and invasion through 14-3-3ε/Rac1 pathway.

Our study further demonstrated that NF45 was able to activate Tiam1-Rac1 signalling. However, the underlying mechanisms through which overexpression of NF45 triggers

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Tiam1 activation are not understood. Next, our data demonstrate that 14-3-3ε was upregulated when NF45 was overexpressed, and 14-3-3ε knockdown eliminated the Tiam1-Rac1 activation triggered by NF45 overexpression. However, the correlation

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between NF45 and 14-3-3ε protein expression needs more clinical data to verify. The next question was how NF45 upregulated the 14-3-3ε expression. Previous studies have demonstrated that NF45 interacts with various RNA-binding proteins (RBPs) which are directly involved in modulating the alternative splicing and stability of specific pre-mRNAs [11, 30]. NF45-NF90 complexes occupy the c-fos enhancer/promoter region and stimulate c-fos gene transcription [31]. So, we hypothesized that NF45 may also stimulate 14-3-3ε expression through the promoter of the 14-3-3ε gene. The fact that NF45 associates with NF90 in the nucleus and regulates IL-2 gene transcription is its best-known function [32], this may also be

ACCEPTED MANUSCRIPT related to the regulation of 14-3-3ε expression. However, it needs detailed experimental data to investigate the relation between IL-2 and 14-3-3ε in the future.

The molecular mechanisms of 14-3-3ε activating Tiam1-Rac1 signalling remain

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obscure. Our previous studies have shown that binding of 14-3-3ζ to Tiam1 could activate the GEF function of Tiam1 and facilitate Rac1 activation [32]. An important question for future studies is whether 14-3-3ε binds to Tiam1 to upregulate Rac1

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activity and whether other moleculars are involved in 14-3-3ε-Tiam1-Rac1 signalling.

In conclusion, this study demonstrates that NF45 can significantly promote ESCC cell

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proliferation and invasion by increasing Rac1 activity through 14-3-3ε protein. The newly identified NF45/14-3-3ε/Tiam1 pathway helps us to further identification of new therapeutic targets for ESCC.

Conflict of interest

Acknowledgments

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The authors declare that they have no conflict of interest.

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This study was supported by a grant from National Natural Science Foundation of

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China (81401323).

Author Contributions Designed the studies: W.S.H., W.J.J. Undertook the experimental work: Y.W.J, T.S., Contributed to figures and manuscript preparation: Y.W.J, T.J, X.K.Y. All authors read and approved the final manuscript.

Ethics approval and consent to participate: The use of human tumor tissue and clinical data was approved by the Human Research Committee of Huazhong University of Science and Technology. All patients gave their written informed consent.

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References: Cancer J Clin 66 (2016) 115-132.

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[1] W. Chen, R. Zheng, P.D. Baade, S. Zhang, H. Zeng, F. Bray, A. Jemal, X.Q. Yu, J. He, CA [2] P. Marcoulatos, E. Avgerinos, D.V. Tsantzalos, N.C. Vamvakopoulos, J Interferon Cytokine Res 18 (1998) 351-355.

[3] T.W. Reichman, L.C. Muniz, M.B. Mathews, MOL CELL BIOL 22 (2002) 343-356.

[4] D. Guan, N. Altan-Bonnet, A.M. Parrott, C.J. Arrigo, Q. Li, M. Khaleduzzaman, H. Li, C.G. Lee,

SC

T. Pe'Ery, M.B. Mathews, MOL CELL BIOL 28 (2008) 4629-4641.

[5] P.N. Kao, L. Chen, G. Brock, J. Ng, J. Kenny, A.J. Smith, B. Corthesy, J BIOL CHEM 269 (1994) 20691-20699. [6] G.N. Barber, RNA BIOL 6 (2009) 35-39.

M AN U

[7] T. Ni, G. Mao, Q. Xue, Y. Liu, B. Chen, X. Cui, L. Lv, L. Jia, Y. Wang, L. Ji, J MOL HISTOL 46 (2015) 325-335.

[8] R.A. Shamanna, M. Hoque, T. Pe'Ery, M.B. Mathews, ONCOGENE 32 (2013) 5176-5185. [9] C. Wan, C. Gong, L. Ji, X. Liu, Y. Wang, L. Wang, M. Shao, L. Yang, S. Fan, Y. Xiao, X. Wang, M. Li, G. Zhou, Y. Zhang, MOL CELL BIOCHEM 410 (2015) 25-35.

[10] T. Higuchi, H. Todaka, Y. Sugiyama, M. Ono, N. Tamaki, E. Hatano, Y. Takezaki, K. Hanazaki, T. Miwa, S. Lai, K. Morisawa, M. Tsuda, T. Taniguchi, S. Sakamoto, J BIOL CHEM 291 (2016)

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21074-21084.

[11] M. Marchesini, Y. Ogoti, E. Fiorini, S.A. Aktas, L. Nezi, M. D'Anca, P. Storti, M.K. Samur, I. Ganan-Gomez, M.T. Fulciniti, N. Mistry, S. Jiang, N. Bao, V. Marchica, A. Neri, C. Bueso-Ramos, C.J. Wu, L. Zhang, H. Liang, X. Peng, N. Giuliani, G. Draetta, K. Clise-Dwyer, H. Kantarjian, N. Munshi, R. Orlowski, G. Garcia-Manero, R.A. DePinho, S. Colla, CANCER CELL 32 (2017) 88-100.

EP

[12] S. Ni, J. Zhu, J. Zhang, S. Zhang, M. Li, R. Ni, J. Liu, H. Qiu, W. Chen, H. Wang, W. Guo, Tumour Biol 36 (2015) 747-756.

[13] Y. Liu, F. Song, W.K. Wu, M. He, L. Zhao, X. Sun, H. Li, Y. Jiang, Y. Yang, K. Peng, CELL

AC C

BIOCHEM FUNCT 30 (2012) 271-278. [14] Y. Kosaka, K.A. Cieslik, L. Li, G. Lezin, C.T. Maguire, Y. Saijoh, K. Toyo-oka, M.J. Gambello, M. Vatta, A. Wynshaw-Boris, A. Baldini, H.J. Yost, L. Brunelli, MOL CELL BIOL 32 (2012) 5089-5102.

[15] T.A. Liu, Y.J. Jan, B.S. Ko, Y.J. Wu, Y.J. Lu, S.M. Liang, C.C. Liu, S.C. Chen, J. Wang, S.K. Shyue, J.Y. Liou, ONCOTARGET 6 (2015) 38967-38982. [16] H. Wang, H. Huang, W. Li, X. Jin, J. Zeng, Y. Liu, Y. Gu, X. Sun, G. Wen, Y. Ding, L. Zhao, J SURG ONCOL 106 (2012) 224-231. [17] K.L. Rossman, Der CJ, J. Sondek, Nat Rev Mol Cell Biol 6 (2005) 167-180. [18] H.K. Bid, R.D. Roberts, P.K. Manchanda, P.J. Houghton, MOL CANCER THER 12 (2013) 1925-1934. [19] T. Song, X. Tian, F. Kai, J. Ke, Z. Wei, L. Jing-Song, W. Si-Hua, W. Jian-Jun, ONCOTARGET 7 (2016) 64260-64273.

ACCEPTED MANUSCRIPT [20] P. Boissier, U. Huynh-Do, CELL SIGNAL 26 (2014) 483-491. [21] S.J. Heasman, A.J. Ridley, Nat Rev Mol Cell Biol 9 (2008) 690-701. [22] H. Kobayashi, Y. Ogura, M. Sawada, R. Nakayama, K. Takano, Y. Minato, Y. Takemoto, E. Tashiro, H. Watanabe, M. Imoto, J BIOL CHEM 286 (2011) 39259-39268. [23] L. Liu, Y. Dai, J. Chen, T. Zeng, Y. Li, L. Chen, Y.H. Zhu, J. Li, Y. Li, S. Ma, D. Xie, Y.F. Yuan, X.Y. Guan, HEPATOLOGY 59 (2014) 531-543. [24] S. Tong, S.C. Chen, K.Y. Xu, B. Fang, S.H. Wang, J.J. Wang, ARCH BIOCHEM BIOPHYS 643

RI PT

(2018) 7-13.

[25] N.X. Xiong, H.Y. Zhao, F.C. Zhang, Z.Q. He, NEUROSCI BULL 23 (2007) 41-45.

[26] C. Lin, L. Song, A. Liu, H. Gong, X. Lin, J. Wu, M. Li, J. Li, ONCOGENE 34 (2015) 384-393.

[27] H. Zhou, M.G. Kann, E.K. Mallory, Y.H. Yang, A. Bugshan, N.O. Binmadi, J.R. Basile, NEOPLASIA 19 (2017) 65-74.

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[28] M.J. van Hemert, H.Y. Steensma, G.P. van Heusden, BIOESSAYS 23 (2001) 936-946.

[29] H. Kobayashi, Y. Ogura, M. Sawada, R. Nakayama, K. Takano, Y. Minato, Y. Takemoto, E. Tashiro, H. Watanabe, M. Imoto, J BIOL CHEM 286 (2011) 39259-39268.

[30] M. Dutertre, S. Lambert, A. Carreira, M. Amor-Gueret, S. Vagner, TRENDS BIOCHEM SCI 39

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(2014) 141-149.

[31] T. Nakadai, A. Fukuda, M. Shimada, K. Nishimura, K. Hisatake, J BIOL CHEM 290 (2015) 26832-26845.

[32] P.N. Kao, L. Chen, G. Brock, J. Ng, J. Kenny, A.J. Smith, B. Corthesy, J BIOL CHEM 269 (1994)

Figure legend

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20691-20699.

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Figure 1. NF45 is upregulated in ESCC and correlated with poor prognosis. A. NF45 protein expression level in 6 paired human non-cancerous esophageal tissues (N) and ESCC tissues (T) was determined by western blotting analysis. B. qPCR analyses of

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NF45 mRNA expression in matched normal tissues and ESCC tissues. C. Representative IHC images showing protein level of NF45 in ESCC specimens and non-cancerous tissues. Scale bar, 100 µm. D. The quantification of IHC is shown as dot blots. The black bars represent the mean. The color bars represent the mean ± standard deviation. (e) Kaplan-Meier DFS curves for ESCC patients. Disease-free survival is defined as length of time after primary treatment for cancer ends that the patient survives without any signs or symptoms of that cancer. E. Kaplan-Meier OS curves for ESCC patients in TCGA database. Overall survival rate is the percentage of people in a study or treatment group who are still alive for a certain period of time

ACCEPTED MANUSCRIPT after they were diagnosed with or started treatment for a disease.

Figure 2. NF45 promotes ESCC cell proliferation. A. Western blot analysis showed the expression of NF45 in normal esophageal epithelial cell line (Het-1A) and ESCC

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cell lines (EC18, ECA109, KYSE180, KYSE150). B. Western blot analysis of NF45 expression in ECA109 (left panel) and KYSE180 (right panel) ESCC cells stably

expressing NF45-shRNA or NF45-cDNA. C. A MTT assay was carried out over a 4-day culture period. Data are presented as the means ± SD of three independent

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experiments. Relative proliferation rate is equal to the value of optical density (OD)

measured by MTT. D. Evaluating the effects of NF45-shRNA or NF45-cDNA on the

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proliferation of ECA109 and KYSE180 cells, respectively, using colony formation assay. Representative images from three independent experiments. E. Images of the tumors incised from the mice injected with NF45-cDNA transfected cells subcutaneously. Weights of tumors are summarized in the right panel. The tumor wights is (0.61 ± 0.27 g) in vector group vs. (1.82 ± 0.34 g) in NF5 group, n = 5. Data

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are shown as averages±SD.

Figure 3. NF45 promotes ESCC cell invasion. A. Representative images of

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wound-healing assays in ECA109 (left panel) and and KYSE180 (right panel) cells. Scale bar 400 µm B. Representative images of transwell invasion assay in ECA109 (left panel) and and KYSE180 (right panel) cells. Data are presented as the means ±

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SD of three independent experiments.

Figure 4. NF45 activates Tiam1-Rac1 signalling and upregulates 14-3-3ε expression. A. Cell lysates from ECA109 and and KYSE180 cells were subjected to a Tiam1 activity assay and Rac activity assay. B. The relative 14-3-3 mRNA was analyzed by qRT-PCR in NF45-cDNA transfected KYSE180 cells compared to control cells. Relative mRNA expression= NF45-cDNA cells target gene (2−∆∆Ct)/ control cells target gene (2−∆∆Ct). Data are presented as the means ± SD of three independent experiments. *P < 0.05 C. The levels of 14-3-3ε protein were determined by western

ACCEPTED MANUSCRIPT blotting in NF45-cDNA transfected KYSE180 cells. D. Representative immunofluorescent staining images showing increased expression of 14-3-3ε in NF45-expressing KYSE180 cells. Scale bar 25 µm E. Analysis showing significant Pearson correlations of NF45 with 14-3-3ε (n = 77) in ESCC tumor samples. R is

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Pearson correlation coefficient. F. The scatter plot shows the correlation of NF45 and 14-3-3ε expression in TCGA esophageal squamous carcinoma database.

Figure 5. 14-3-3ε is involved in NF45-induced Rac1 activation in the process of

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ESCC cell invasion. A. Transient knockdown of 14-3-3ε in KYSE180 cell lines. B.

MTT assay showing that 14-3-3ε shRNA could inhibit NF45-induced cell growth. C.

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Colony formation assay showing that 14-3-3ε shRNA could inhibit NF45-induced cell growth. D. Images of the xenografted tumors formed in mice injected with infected KYSE180 cells. Weights of tumors are summarized in the right panel. The tumor weights are (0.37 ± 0.21 g) in vector group , (1.35 ± 0.31 g)g in NF5 group, and (0.31 ± 0.19 g) in NF5+sh14-3-3ε group. Data are shown as averages±SD. E. Evaluating the

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effects of 14-3-3ε shRNA on the NF45-induced cell migration of KYSE180 cells using wound-healing assays. Scale bar 400 µm F. Cell invasion was evaluated by using transwell invasion assay. Data are presented as the means ± SD of three

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independent experiments. G. Tiam1 activity and Rac1 activity assay showing that

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14-3-3ε shRNA could inhibit NF45-induced Tiam/ Rac1 activation.

ACCEPTED MANUSCRIPT Table 1. The association between clinical parameters with NF45 mRNA

Gender Male Female Age ≤60 >60 Smoking status Nonsmokers Smokers Differentiation

Total No.

NF45

P-value

Low(n=39)

High(n=38)

44 33

24 15

20 18

0.57

41 36

15 24

26 12

0.01

30 47

14 25

16 22

0.74

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0.28

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Features

Well Moderate

27 39

17 17

10 22

Poor Tumor invasion

11

5

31 46

21 18

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10 28

0.02

0.54

44 33 36 41

21 18

23 15

20 19

16 22

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T1/T2 T3/T4 Lymph node † metastasis N0 N1 † Stage Ⅰ/ⅡA ⅡB/Ⅲ

6

0.46

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† The TNM classification is from 8th Edition of the AJCC Cancer Staging Manual: Esophagus and Esophagogastric Junction

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