Introduction
Renal cell carcinoma (RCC) is a malignant cancer of the tubular cells of the kidney, which accounts for 2–3% of adult malignant tumors and its morbidity and mortality are still increasing [
1]. Although most incidentally detected lesions are small low-grade tumors, up to 17% of all renal cell carcinomas have distant metastases at the time of diagnosis [
2]. However, there is no effective treatment for metastatic renal cell carcinoma. Interferon-α (INF-α) and interleukin-2 (IL-2), which were the standard for the treatment of metastatic RCC, fail to provide satisfactory clinical benefit because of their known significant toxicity and modest overall response rates (ORRs) [
3]. Molecular-targeted therapy is one of the major strategies for the treatment of metastatic RCC. Several molecular-targeted therapies such as the mTOR inhibitor temsirolimus and the VEGF inhibitor bevacizumab have been proved to improve the clinical outcomes of RCC patients [
4,
5]. Because of the complexity of the pathogenesis of malignant tumors, molecular-targeted therapies still have the limitations including low efficiency and drug resistance [
6]. In the past decades, although several breakthroughs have been achieved in the study of RCC, the underlying mechanisms of metastasis in renal cell carcinoma remain unclear. Therefore, it is important to explore the mechanism of metastasis and find a new molecular target for RCC.
CEP55 has been initially identified as an important component of abscission, participating in the separation of two daughter cells [
7]. Currently, it has been found that CEP55 serves a pivotal role in multiple cancers, such as oral squamous cell carcinoma and breast cancer [
8,
9]. Previous studies reported that neoplasms with high levels of CEP55 are typically associated with patients’ clinical characteristics and poor outcomes. CEP55 not only could promote the proliferation of cancer cells, but also promotes invasion and metastasis via the increasing of FOXM1 and the activation of MMP-2 in oral squamous cell carcinoma [
9,
10]. However, the detailed mechanisms of tumorigenesis and clinical outcomes of CEP55 overexpression in renal cell carcinoma remain unclear.
Epithelial–mesenchymal transition (EMT) is initially discovered in embryonic development, a shift from epithelial states toward mesenchymal states, accompanying the alteration of adhesion molecules expressed on the cell surfaces [
11]. E-cadherin is the most commonly used marker for the epithelial trait and N-cadherin is used for the mesenchymal trait [
12]. Many researches reveal that EMT is one of the important cellular phenomena that promotes tumor metastasis. Tumor cells bearing low epithelial phenotype and high mesenchymal phenotype have been referred to more flexible and less stable, with the tendency toward metastasis [
13]. Snail, slug and zinc finger E-box binding homeobox 1 (ZEB1) are the key transcription factors that could induce EMT by regulating E-cadherin and N-cadherin expression directly or indirectly [
14]. In our study, we found that CEP55 could regulate the process of EMT in RCC. Furthermore, CEP55 promoted cell migration and invasion by activation of PI3KAKT/mTOR pathway. These results suggest a novel CEP55 function in metastasis and might serve as a potential therapeutic target for RCC.
Materials and methods
Patient tissue samples
The RCC tissues and adjacent normal tissues were obtained from patients who underwent surgery only in Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University. Five paired samples were used for mRNA microarray analysis and ten paired samples were used for validation. RCC tissues from 57 patients were used for IHC staining.
IHC staining
IHC staining was performed using a Dako Envision System (Dako, Santa Clara, CA, USA) following the manufacturer’s protocol. Sections were blocked using serum-free protein block buffer (Dako, Santa Clara, CA, USA) for 30 min, after which they were incubated with anti-CEP55 (1:200, Atlas Antibodies, Stockholm, Sweden). The tissue sections were graded as high expression when 10% of tumor cells showed immunopositivity. Biopsies with less than 10% tumor cells showing immunostaining were considered low expression.
Cell culture
The human renal cell carcinoma cell lines ACHN and 786-O were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), which were cultured in RPMI 1640 culture media supplement (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Sigma, St. Louis, MO, USA). The cancer cell lines were cultured at 37 °C in a humidified atmosphere of 5% CO2.
Microarray analysis
5 paired samples were used for microarray work, which were performed by CapitalBio Technology, China. Hybridized with the Agilent human mRNAs Array V2.0, more than 29,000 mRNA were detected. A fold change of ≥ 2.0 and P ≤ 0.05 was set as the threshold for statistically significant up and downregulated genes, respectively.
RNA interference and cell transfection
siRNAs targeting CEP55 (si#1: 5′-GGGAGAAATTGCACACTTAtt-3′; si#2: 5′-GGACTTTTAGCAAAGATCTtt-3′ [
9]) were constructed by Genepharma (Suzhou, China). Lipofectamine RNAiMAX (Thermo Fisher Scientific, MA, USA) was used for transient transfection. All functional assays were conducted 48 h after siRNA transfection. Two types of lentivirus plasmids Lv-CEP55 and Lv-vector (blank vector), conducted from Genechem (Shanghai, China) were transfected into RCC cells to establish the overexpressed group and the control group.
MTS assay
The cell proliferation was measured by MTS assay (CellTiter 96® AQueous one Solution Cell proliferation Assay, Promega, Madison, WI, USA). 1 × 103 cells per well were seeded into 96-well plates. 20 μl MTS solution was added into each well and then incubated at 37 °C, protected from light for 4 h. Optical density was measured at 490 nm. The assay was conducted at 0 h, 24 h, 48 h, 72 h, 96 h and 120 h to generate a growth curve.
1 × 103 cells per well were seeded into six-well plates and cultured for 2 weeks to form optimal colonies. Colonies were fixed by paraformaldehyde, stained by crystal violet and then photographed.
Wound healing assay
Cells were seeded in six-well plates and cultured to produce a confluent monolayer. Wound areas were scraped using 200-μl pipette tips. Washed three times with PBS to remove debris and then adding RPMI 1640 culture media without FBS. Wound closure was observed and photographed at 0–24 h under an inverted microscope.
Migration and invasion assay
In migration assay, 1 × 105 cells were suspended in 200 μl serum-free medium for per well and seeded into the upper chamber and RPMI 1640 culture media with 10% FBS was added into the lower chamber. In invasion assay, 1 × 105 cells suspended in 200 μl serum-free medium were seeded into the Matrigel-coated upper chambers. Migratory cells through the polycarbonate membrane after 6 h, and invasive cells after 48 h were fixed by paraformaldehyde, stained by crystal violet and counted at 100 × magnification in at least five randomly selected fields.
Real-time quantitative reverse transcription PCR (qRT-PCR) analysis
Total RNA was isolated from cells by RNAiso Plus (Takara, Kusatsu, Japan). Reverse transcription reaction was carried out with the program for 15 min at 37 °C, and then for 5 s at 85 °C. Quantification of CEP55 and GAPDH was performed with the SYBR® PrimeScript™ RT-PCR Kit (Takara, Kusatsu, Japan). The following primer sets for CEPEE and GAPDH were used: CEP55 primers forward: 5′-TTGGAACAACAGATGCAGGC-3′, reverse: 5′-GAGTGCAGCAGTGGGACTTT-3′; GAPDH primers forward: 5′-GGGAGCCAAAAGGGTCAT-3′, reverse: 5′-GAGTCCTTCCACGATACCAA-3′. Relative quantification was performed with the 2−∆∆Ct method.
Western blot analysis
Total protein was isolated from cells by RIPA lysis buffer and separated by polyacrylamide gels, and then transferred onto PVDF membranes. After blocked with 5% BSA solution, the membranes were incubated with primary antibodies, including CEP55, E-cadherin, N-cadherin, ZEB1 (1:1000, Santa Cruz Biotechnology, CA, USA), PI3K, and mTOR Pathway Antibody Sampler Kit (1:1000, CST, Boston, MA, USA). Membranes were incubated with secondary antibodies for 1 h at room temperature. Protein bands were treated with chemiluminescent HRP Substrate (Millipore Corporation, Billerica, MA, USA) and photographed.
Data downloaded from the cancer genome atlas (TCGA)
Data of CEP55 expression and corresponding clinical information for 532 RCC patients from TCGA database were downloaded from cBioPortal (
http://www.cbioportal.org/ [
15,
16]), provided visualization, analysis and download of large-scale cancer genomics data sets.
Statistical analysis
Statistical analysis was done by the software SPSS 18.0.0. Quantitative variables are presented as the mean ± SD. The significance of the quantitative PCR results from patient tissues and cell lines, the western blot results measured by ImageJ, and the vitro assays were determined by Student’s t test (two-tailed). The significance of clinical characteristics was determined by t test and χ2 test. The Kaplan–Meier method and log-rank test were used to evaluate the correlation between CEP55 expression and patients’ survival. P < 0.05 was considered statistically significant.
Discussion
Previous studies have shown that CEP55 is aberrantly expressed in multiple types of cancers, and involved in cancer progression, including proliferation, migration and invasion [
18]. CEP55 knockdown could suppress cellular proliferation as a result of cell cycle arrest at the G2/M phase in gastric cancer [
19]. In oral cavity squamous cell carcinoma, CEP55 could promote MMP-2 expression and activation through upregulation of FOXM1, serving as a driver of invasion and metastasis [
9]. In this study, we investigated the expression level of CEP55 in RCC tissues and adjacent normal tissues, and a panel of RCC cell lines. The expression of CEP55 mRNA was significantly associated with poor survival and advanced features of patients with RCC, including neoplasm disease stage, histologic grade and TNM stage. The results suggested that CEP55 might function as an oncogene in RCC. To elucidate whether CEP55 affects the biological behavior of RCC cells, we designed a series of experiments to evaluate the biological function of CEP55. Then we found that knockdown of CEP55 could significantly decrease the proliferation, migration and invasion of RCC cells. In contrast, overexpression of CEP55 enhanced the capacity of migration and invasion in RCC cells.
Emerging evidence has shown that EMT is a critical component of the metastatic process, leading the dissociation of connected cells from the primary tumor and their intravasation into blood vessels [
20,
21]. Loss of epithelial marker expression and gain of the mesenchymal marker expression are the hallmarks of the EMT process. EMT-TF such as snail, slug and ZEB1 are the spotlight of EMT investigation [
13]. Upregulation of ZEB1 has been observed in various cancers and the expression of ZEB1 is significantly correlated to tumor grade and lymphatic or distant metastasis [
22‐
24]. Previous studies showed that ZEB1 induces EMT by binding to the promoter of E-cadherin and depressing the expression level of E-cadherin. In addition, it is known that miRNA-200 acts as a regulator of E-cadherin and plays a crucial role in modulating EMT in RCC. ZEB1 has also been proven to inhibit E-cadherin by forming an important negative feedback loop with miRNA-200 [
25,
26]. In the present study, our data indicated that knockdown of CEP55 could increase the expression of E-cadherin and decrease the expression of N-cadherin and ZEB1. In addition, after upregulation of CEP55 expression, the expression of N-cadherin and ZEB1 was increased and E-cadherin was decreased in RCC cells. These findings suggest that CEP55 may be an EMT inducer and has potential as a novel molecular target for anti-metastasis therapy in RCC.
Increasing evidence shows that the activation of PI3K/AKT/mTOR pathway is crucial for EMT process [
27,
28]. Mechanistically, activated PI3K could phosphorylate phosphatidylinositol 4, 5-bisphosphate (PIP2), which would bind and phosphorylate AKT [
29]. Furthermore, phosphorylation of AKT could activate mTOR to control motility, invasion and phenotype transformation of cells and contribute to EMT [
30]. Previous study showed that miR-200c suppresses the expression of ZEB1 through PI3K/AKT signaling pathway in non-small cell lung cancer [
31]. Furthermore, Src promotes the process of EMT by upregulation of ZEB1 and ZEB2 through AKT signaling pathway in gastric cancer cells [
32]. In addition, it has been reported that CEP55 binds to PI3K and increases the stability of this subunit, resulting in increased AKT activation as observed by an increase in S473 phosphorylation [
33]. In our study, we found that activation of PI3K/AKT/mTOR pathway was positively associated with the expression of N-cadherin and ZEB1 and negatively associated with the expression of E-cadherin. In addition, after inhibiting PI3K with LY294002, the expression of E-cadherin was upregulated while N-cadherin and ZEB1 was downregulated. Therefore, it came to the conclusion that CEP55 activated the PI3K/AKT/mTOR pathway and participated in EMT process of RCC.
At present, molecular target therapy is the first option for the management of advanced RCC. Multitarget tyrosine kinase inhibitors (TKIs) which against the VEGF receptors (VEGFRs), PDGF receptors (PDGFRs) have already been shown to improve survival of RCC patients [
34]. A randomized, placebo-controlled, phase 3 study showed that mTOR inhibitor everolimus could improve the median progression-free survival (PFS) of advanced RCC patients [
35]. However, the toxicity and resistance has limited their clinical application. Activation of alternative or compensatory pathway of VEGFR and mTOR signaling contributes to the upregulation of HIF, which leads to the resistance to VEGFR- and mTOR-targeted therapies [
36]. Due to the negative feedback loop between mTOR, PI3K and MAPK, inhibition of mTOR would lead to the activation of PI3K/AKT and MAPK pathway [
37]. In our study, CEP55 inhibition could simultaneously weaken PI3K/AKT/mTOR pathway, so CEP55 may be an effective target for metastasis RCC treatment.
Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (Grant nos. 81472388, 81672534), supported by grant [2013]163 from Key Laboratory of Malignant Tumor Molecular Mechanism and Translational Medicine of Guangzhou Bureau of Science and Information Technology, supported by Grant KLB09001 from the Key Laboratory of Malignant Tumor Gene Regulation and Target Therapy of Guangdong Higher Education Institutes, supported by grant from Guangdong Science and Technology Department (2013B021800110, 2015B050501004), supported by Guangdong Natural Science Foundation (2014A030310133).
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