ERRα promotes gallbladder cancer development via activating PI3K/AKT signaling pathway mediated by Nectin-4

Background: Gallbladder cancer (GBC) is the most common malignant tumour of the bile duct with a poor prognosis. The estrogen-related receptor alpha (ERRα) is a nuclear receptor that has been associated with metabolic processes and cancer progression. Increased ERRα has been shown in endocrine-related cancer and non-endocrine-related cancer. Nevertheless, its role in GBC remains unknown. Methods: ERRα expression was analyzed by immunohistochemistry in 59 GBC samples. Its association with clinicopathologic characteristics was evaluated. The effect of ERRα on GBC cell proliferation and invasion was evaluated by loss- or gain-of-function assays in vitro and in vivo. The influence of ERRα on EMT biomarkers, Nectin-4 and PI3K/AKT signaling pathway was detected by western blotting. Inhibition of Nectin-4 was conducted to explore the potential mechanism of ERRα in GBC. Results: Our study reveals increased ERRα expression in GBC tissues vs. non-tumour adjacent tissues. ERRα expression is significantly positively correlated with high TNM stage, high invasion depth and lymph node metastasis, while negatively associated with prognosis. Targeted depletion of ERRα by lentivirus-mediated shRNA decreased cell proliferation in vitro and in vivo. ERRα promoted cancer cell migration and epithelial-mesenchymal transition by regulating the expression of EMT relevant genes. Ectopic expression of ERRα promoted GBC cell growth and invasion in vitro, while inhibiton of Nectin-4 attenuated cell growth and invasion induced by ERRα. Moreover, ERRα overexpression activated the PI3K/AKT signaling pathway while this effect can be blocked by Nectin-4 depletion. Nectin-4 was involved in the oncogenic function of ERRα to activate PI3K/AKT signaling pathway in GBC. Conclusions: ERRα promotes GBC progression via activating PI3K/AKT signaling pathway mediated by Nectin-4. ERRα may be a potential prognostic factor and molecular therapeutic target for GBC.


Background
Gallbladder cancer (GBC) is the most common biliary tract malignancy with an increasing incidence worldwide [1,2] . Due to easier local infiltration and distant metastasis, gallbladder carcinoma has limited therapeutic options and a poor outcome with conventional therapy [3][4][5] . An increasing need for novel prognostic biomarkers and targeted therapeutics based on epidemiology exists. The female-to-male (F/M) ratio of GC incidence rates varied greatly: it exceeded 5 in several high-risk areas as well as in lowrisk areas, but was typically between 2 and 3 [6,7] . Therefore, we investigated several sexspecific genes and found that Estrogen-related receptor α(ERRα) promotes human gallbladder tumor growth.
ERRα is structurally most related to the canonical estrogen receptor(ER) [8,9] . Unlike ERs, ERRα does not bind to naturally occurring estrogens, but functions by binding to estrogenrelated responsive element(ERE) and estrogen-related responsive element (ERRE) , thus regulating bioenergetic pathways required for cell-and tissue-specific functions [10] . ERRα is one of the orphan members of the nuclear receptor superfamily, and, as such, acts a transcriptional regulator [11] . In contrast to several other members of the NR family, no natural ligand has been, to date, identified for ERRα, which is thus referred to as "orphan" [12] . This receptor promotes cellular proliferation, migration, invasion, resistance to hypoxia, as well as metabolic reprograming, which all contribute to cancer aggressiveness [11,[13][14][15] . However, the expression and function of ERRα in gallbladder cancer remains unclear.
In this study, we demonstrated for the first time that a significant correlation exists between higher expression of ERRα and a worse prognosis in GBC patients. We showed 4 that shRNA-mediated knockdown of ERRα markedly reduced gallbladder cancer cell growth and tumorigenicity in vitro an in vivo. And ERRα promoted GBC cell migration and invasion by inducing epithelial-mesenchymal transition (EMT). Conversely, upregulating ERRα promoted the migration and invasion ability of GBC cells. We also demonstrated that the Nectin-4 and PI3k/AKT pathway is involved in the oncogenic effects of ERRα in GBC cells.

Quantitative real-time PCR
Trizol reagent (Takara) was used to extract total RNA from cells. cDNA was synthesized by

Immunofluorescence analysis
Cells were fixed in 4% paraformaldehyde and permeabilized in 0.5% Triton X-100 at room temperature. After blocking with 1% BSA for 1 h, the cells were incubated with anti-ERRα 6 antibody at 4 °C overnight. Then, the cells were incubated for 30 min at 37 °C with Goat Anti-Rabbit IgG ( Proteintech, Wuhan, China)and counterstained with DAPI.

RNA interference, construction of plasmids and transfection
The short hairpin RNA(shRNA) sequence targeting human ERRα was 5'-GCGAGAGGAGUAUGUUCUA-3'. The sequence of negative control shRNA was 5'-TTCTCCGAACGTGTCACGT-3'. Both shRNAs were synthesized and inserted into the PGMLV-SC5 lentivirus core vector, containing a cytomegalovirus(CMV)-driven enhanced green fluorescent protein (EGFP) reporter gene. These lentiviruses were constructed by Genomeditech (Shanghai, China). NOZ and OCUG cells were infected with concentrated virus in serum-free medium. The supernatant was replaced with complete culture medium after 24 h. ERRα expression in the infected cells was validated by qRT-PCR and western blot assays after 120 h.

Construction of plasmids and transfection
The full-length sequence of ERRα(GenBank accession number NM_004451.4) was cloned into the pCDNA3.1 expression vector (Genomeditech, Shanghai, China) and transfected into GBC-SD cells using ViaFect™ Transfection Reagent following the manufacturer's instructions (Promega). Stable ERRα-expressing clones were selected for 2 weeks using neomycin (Genechem), and the expression level of ERRα was determined by western blot assays. Empty vector-transfected cells (MOCK) were used as control.

Antibodies and western blotting
The anti-ERRα antibody was purchased from Novus Biologicals, Canada. The anti-Nectin-4 antibody were purchased from Abcam (Cambridge, MA, USA). The anti-Lumin B1 antibody were purchased from Boster (California, USA). Anti-E-cadherin, -vimentin, -PI3K, -p-PI3K (Tyr607), -AKT, -p-AKT (Ser473), and β-actin antibodies were acquired from Cell Signaling Technology (Danvers). Proteins from cells were separated by SDS-PAGE and then transferred onto PVDF membranes (Millipore Corp., Billerica, MA, USA). The membranes were blocked in 5% skimmed milk(5% BSA for p-PI3K and p-AKT) and then incubated with at 4° C overnight with primary antibodies. After washing, the membranes were reacted with secondary antibody at room temperature. The blots were detected by enhanced chemiluminescence (Millipore, Billerica, MA). β-Actin and Lumin B1 were used as the loading controls for total protein and nucleoprotein respectively. All assays were performed in triplicates.

In vitro tumorigenesis assays
A Cell Counting Kit-8 (CCK-8; Dojindo) cell proliferation assay was performed to assess the proliferation of cells according to the manufacturer's instructions. The absorbance value was measured at 450 nm. A colony formation assay was performed to evaluate the anchorage-independent growth of NOZ, OCUG and GBC-SD cells. After culturing for 9 days, cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet (Sigma) for 15 min. The numbers of colonies (>50 cells/colony) were photographed and counted.

In vitro migration and invasion assays
Cell migration and invasion were performed using 8-μm transwell filters (BD Biosciences,

Nude mice subcutaneous xenograft model
Ten Nude nu/nu mice (3-5 weeks, 12-16g) were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China). These mice were randomly divided into two groups (Lv-shNC and Lv-shERRα). 2×10 6 NOZ cells were subcutaneously injected into the left axilla of each mouse. Tumor size was monitored every week. Four weeks later, all the mice were executed by dislocation of the cervical vertebra and all implanted tumors were surgically collected. Tumor volume was calculated using the following formula: tumor volume (mm 3 ) = 1/2 × width 2 × length, where the width and length were the shortest and longest diameters, respectively. All of the experiments were carried out followed by the protocols of the NJMU Institutional Animal Care and Use Committee.

Statistical analysis
IBM SPSS Statistics 19.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analyses.
The quantitative data were expressed as the mean ± standard deviation (SD). The independent Student's t-test was used to compare the means of two groups. The difference between ERRα expression and clinicopathologic parameters were performed using χ2 tests. The Kaplan-Meier test was performed for univariate survival analysis.
Multivariate analysis was determined by using the Cox proportional hazards model.
Significant differences between groups were considered for p < 0.05. All of the results are representative of three independent experiments.

Results
Nuclear ERRα were highly expressed and associated with a poor outcome in GBC patients 9 To explore the role of ERRα in GBC progression, we tested nuclear ERRα expression in tumor samples from patients with GBC or cholelithiasis by using IHC (Fig. 1a) . 67.80% (40/59) of the GBC patients showed positive nuclear ERRα staining (Table 1). ERRα expression level was significantly higher in tumor tissues than in cholelithiasis tissues (Fig. 1b, P<0.01). A Clinicopathological correlation analysis of the 59 GBCs found that higher ERRα levels were associated with higher AJCC TNM stage(P<0.01), deeper invasion depth(P<0.01) and lymph node metastasis(P<0.01) ( Table 1). More intriguingly, Kaplan-Meier analysis for relapse-free survival(RFS) and overall survival(OS) indicated that positivity of ERRα correlated with shorter RFS and OS( Fig. 1c and 1d, P<0.01). Multivariate Cox regression analysis further revealed that ERRα expression was negatively correlated with post-operative survival( Table 2, P<0.05), indicating ERRα expression level is an independent prognostic marker in GBC patients.

ERRα expression in GBC cell lines
Immunocytochemical staining revealed that ERRα protein expressed in the nucleus of GBC cells (Fig. 2a). We detected the expression of ERRα in four GBC cell lines by RT-PCR and found that ERRα expression was overexpressed in NOZ and OCUG cells and gradually decreased in EH-GB1 and GBC-SD cells (Fig. 2b). Therefore, we chose high ERRα expression NOZ and OCUG cell lines to explore oncogenic function with Lv-shERRα and Lv-shNC.
Conversely, low ERRα expression GBC-SD cell line was chosen to investigate oncogenic function with ERRα-expression vector and empty vector. The efficiencies of lentiviral transduction were examined by EGFP expression under a microscope at 72 h after transduction. Lentiviral transduction efficiencies in these cell lines were higher than 95% (Fig. 2c). Western blotting were performed to confirm transfection efficiency, indicating that ERRα expression was effectively suppressed by Lv-shERRα (Fig. 2d) and overexpressed by ERRα-expression vector (Fig. 2d).

Effects of ERRα knockdown and overexpression on GBC cell proliferation in vitro and in vivo
CCK-8 and colony formation assays were performed to assess whether ERRα affects GBC cell proliferation. As shown in Fig 3a,

the viability of NOZ and OCUG cells tranfected with
Lv-shERRα was significantly decreased compared with negative controls . Fig 3b revealed that the colony formation ability of NOZ and OCUG cells was significantly inhibited by

ERRα promotes GBC cell migration and invasion by inducing EMT
Transwell migration and matrigel invasion assays were performed to assess whether ERRα affects GBC metastasis. As shown in Fig 4a,  through EMT processes, we detected EMT biomarkers by western blotting. After ERRα was depleted, NOZ cells expressed high levels of E-cadherin, which is characteristic of epithelial cells (Fig. 4B). However, the expression level of Vimentin (indicating a mesenchymal phenotype) was lower in ERRα-depleted NOZ cells (Fig. 4B). This indicates 11 that ERRα promotes GBC cell migration and invasion by inducing EMT.

ERRα enhances GBC cell proliferation and migration via activating PI3K/AKT signaling pathway
As shown in Fig. 5g, in NOZ cell line, knockdown of ERRα downregulated the expression of p-PI3K and p-AKT while the expression levels of total PI3K and AKT were not changed.
Conversely, compared to empty vector-transfected cells, overexpression of ERRα in GBC-SD cell line upregulated the expression of p-PI3K and p-AKT. Those results indicated that PI3K/AKT signaling pathway mediated the carcinogenesis of ERRα in gallbladder cancer.

ERRα activated PI3K/AKT signaling pathway by regulating Nectin-4
In our previous study, we have demonstrated that Nectin-4 promotes GBC cell proliferation and migration by activating PI3k/AKT signaling pathway. In the present study, we observed decreased Nectin-4 level in the NOZ cells of ERRα depletion and increased Nectin-4 level in the GBC-SD cells of ERRα overexpression (Fig. 5g). Inhibiton of Nectin-4 attenuated cell growth and invasion induced by ERRα overexpression in GBC-SD cells (Figure 5d, 5e, 5f).

Discussion
Gallbladder cancer is a relatively rare neoplasm with a poor prognosis. In China, the incidence of GBC rises year after year [6] . Late diagnosis and deprived prognosis are major problems for treatment of gallbladder carcinoma. The interplay of genetic susceptibility, lifestyle factors and infections in gallbladder carcinogenesis is still poorly understood.
GBC is several-fold more common in women than in men [7] . However, estrogen-related receptor α(ERRa) has not been linked to GBC.
The orphan receptor estrogen-related receptor α(ERRa) is a member of the nuclear receptor superfamily of ligand-regulated transcription factors. ERRa is structurally most related to the canonical estrogen receptor and has been shown to modulate estrogen signaling in breast cancer [9] . Unlike ERs, ERRα does not bind to naturally occurring estrogens, but functions by binding to estrogen-related responsive element(ERE) and estrogen-related responsive element (ERRE) [10] . ERRα has also been proved to be involved in the carcinogenesis of ERRα in human oral squamous cell cancer [17] , human prostate cancer [18] and human colon cancer [11] . Several mechanisms have been proposed to account for the carcinogenesis of ERRα in different tumors. Kressler D et al demonstrated that the activity of ERRα is regulated by the expression level and activity of its obligate coregulators, peroxisome proliferator activated receptor gamma coactivator-1 alpha and beta (PGC-1α or PGC-1β) [19,20] . By a chemical genomic approach, Chang CY et al found that activation of the Her2/IGF-1 signaling pathways and subsequent C-MYC stabilization upregulate the expression of peroxisome proliferator-activated receptor gamma coactivator-1 beta (PGC-1β), thus upregulating the activation of ERRα in breast cancer [21] .
ERRα has been shown to transcriptionally regulate the expression of several genes, such as WNT11, CCNE1, OPN and OPG, involved in cell cycle, metastasis and metabolism [22] . In the present study, we validated that the high expression of ERRα in GBC tissues correlated with high TNM stage, high invasion depth and lymph node metastasis. ERRα was further identified as an unfavorable independent prognostic factor for GBC, indicating that ERRα might play a role in GBC development.
In the present study, a series of assays in vitro and in vivo showed that downregulating ERRα repressed GBC cell growth and colony formation capability, indicating its role in GBC cell proliferation. Conversely, upregulating ERRα promoted the proliferation of GBC cells.
Moreover, GBC cell migration and invasion ability were inhibited by ERRα knockdown.
Conversely, upregulating ERRα promoted the migration and invasion ability of GBC cells, suggesting that ERRα might be involved in EMT. EMT plays a vital role in cancer invasion and metastasis. During this process, epithelial cells lose their properties and acquire mesenchymal phenotypes. Mesenchymal phenotype cells exhibit increased expression of mesenchymal-related markers, such as vimentin, and decreased expression of epithelialrelated markers such as E-cadherin [23] . In our study, knockdown of ERRα induced EMT by increasing epithelial marker E-cadherin and derceasing mesenchymal marker Vimentin, indicating that ERRα promotes GBC metastasis by inducing EMT. The above results indicate ERRα could serve as a potential diagnostic biomarker and therapeutic target in GBC patients. To get greater insights into the mechanisms of the pro-oncogenic function of ERRα, we found that the protein expressions of Nectin-4 and PI3K/AKT signaling pathway were reduced after downregulating ERRα.
Nectin-4 belongs to the nectin family which has various physiological and pathological functions in humans. Nectin-4 plays a role in the formation and maintenance of adherens junctions in cooperation with cadherins [24][25][26][27] . Other scholars have revealed that nectin-4 is re-expressed as a tumour-associated antigen with pro-oncogenic properties in various carcinomas [28][29][30]. In our previous study [31] , Nectin-4 is critical for GBC proliferation and invasion via activating PI3K/AKT pathway. In the present study, we found that the expression of Nectin-4 was elevated after overexpressing ERRα in GBC cells. Inhibiton of Nectin-4 attenuated cell growth and invasion induced by ERRα upregulation in GBC cells.
Moreover, Nectin-4 depletion blocked ERRα-induced activation of the PI3K/AKT signaling pathway. We hypothesize that Nectin-4 was involved in the pro-oncogenic function of ERRα to activate PI3K/AKT signaling pathway in GBC, thus forming ERRα-Nectin-4-PI3K/AKT axis to promote the growth and invasion of GBC. This axis could be a new promising prognostic biomarker and specific therapeutic target.
As a member of the nuclear receptor superfamily of transcription factors, ERRα plays its role by regulating the transcription of targeted genes. It functions by binding to estrogenrelated responsive element(ERE) and estrogen-related responsive element (ERRE).
Bioinformatics analysis reveals that representative ERE exists in the promotor region of Nectin-4. Taken together, ERRα may regulate the transcription of Nectin-4. For the time to come, dual-luciferase reporter assay system would be used to evaluate the effect of ERRα on the transcription activity of Nectin-4.

Conclusion
This study uncovers for the first time that ERRα enhances GBC cell proliferation and invasion both in vitro and in vivo. In GBC cells, ERRα upregulates Nectin-4 to activate the PI3K/AKT signaling pathway. ERRα-Nectin-4-PI3K/AKT axis may be a potential prognostic factor and molecular therapeutic target for GBC.

Ethics approval and consent to participate
This study was approved by the ethics committee of Nanjing Medical University and written informed consent was obtained from all patients. All the animal experiments were approved by Nanjing Medical University (NJMU) Institutional Animal Care and Use Committee.

Consent for publication
Not applicable.   The role of Nectin-4 in ERRα-induced PI3K/AKT activation and GBC cell proliferation and migration. a-c CCK-8 proliferation, colony formation, migration and invasion assays were conducted to detect the influence of ERRα overexpression on GBC cell proliferation and invasion. d-f The GBC-SD cells overexpressing ERRα were cotransfected with Nectin-4, and the cell grow rate, colony formation and metastasis ability were examined. g NOZ and GBC-SD cells were treated as indicated. ERRα, Nectin-4, Phosphorylated PI3K (Tyr607)/total PI3K, Phosphorylated AKT (Ser473)/total AKT were determined by western blot.
LuminB1 and β-actin were used as the loading controls.