Estrogen-Related Receptor-α Promotes Pancreatic Cancer Progression by Enhancing the Transcription of PAI1 and Activating the MEK/ERK Signaling Pathway

Background: Estrogen-related receptor alpha (ERRα), an orphan nuclear receptor, was reported to be highly associated with the progression and tumorigenesis of several human malignancies. However, the biological role and underlying molecular mechanisms of ERRα in pancreatic cancer (PC) remain unknown. Methods: The expression of ERRα in PC tissues was determined by qRT-PCR and immunohistochemistry. A series of in vitro and in vivo assays were performed to investigate the function of ERRα and Plasminogen activator inhibitor 1 (PAI1) in tumorigenesis in PC cells. The relationship between ERRα and PAI1 was identied by RNA sequencing, Chromatin immunoprecipitation and dual-luciferase reporter assays. The effects of ERRα on the MEK/ERK signaling pathway were determined by western blotting and rescue assays using ERK inhibitor GDC-0994. Results: ERRα was signicantly overexpressed in PC tissues and cell lines. Its high expression was correlated with tumor size, distant metastasis, TNM stage, tumor differentiation and poor prognosis of PC. Subsequent functional assays showed that ERRα promoted PC cell proliferation, tumor growth, as well as migration and invasion via activating the epithelial-mesenchymal transition. In addition, knockdown of ERRα induced apoptosis and G0/G1 cell cycle arrest in PC cells. PAI1 was identied by RNA sequencing, knockdown of which could suppress the cell proliferation, migration and invasion that promoted by ERRα overexpression. Further mechanistic investigation using chromatin immunoprecipitation and dual-luciferase reporter assays revealed that ERRα could bind to the PAI1 promoter region and transcriptionally enhance PAI1 expression. Moreover, our data indicated that ERRα played its oncogenic role in PC via activating the MEK/ERK pathway. Conclusions: Our study demonstrates that ERRα promotes PC progression by enhancing the transcription of PAI1 and activation of the MEK/ERK pathway, pointing to ERRα as a novel diagnostic and therapeutic target for PC. protein expression of cytochrome c, cleaved-caspase 3, cleaved-PARP while downregulated that of bcl-2, suggesting that intrinsic pathway was involved in ERRα knockdown induced cell apoptosis. Aberrant function of cell cycle regulators lead to deregulation of cell cycle progression that result in uncontrolled cell proliferation. The cell cycle analysis demonstrated that ERRα knockdown signicantly decreased the proportion of S phase cells and increased the G0/G1 phase cells. Consistent with the cell cycle analysis, the western blot results showed that the expression of CDK2, CDK4, cyclin D1 was downregulated while p21 Waf1/Cip1 and p27 Kip1 was upregulated after ERRα knockdown, indicating the cell cycle arrest at G0/G1 phase. These data validate a crucial role for ERRα in PC growth. of PC patients. It can promote cell proliferation, migration and invasion via promoting the transcription of PAI1 and activating the MEK/ERK pathway. Our study provides the rst evidence that ERRα might be a potential diagnostic and therapeutic target for PC. blot related markers. C, After silencing of ERRα, ow cytometry showed that the cell cycles of PaTu8988 and PANC1 cells were arrested at G0/G1 phase. D, Cell cycle related proteins were analyzed by western blotting. The expression levels of cleaved-PARP, cleaved-caspase 3, cyclin D1 and p27 Kip1 were detected by IF assay. *p < 0.05, **p < 0.01, ***p < 0.001.


Background
Pancreatic cancer (PC) is one of the most lethal malignant tumors and the fourth leading cause of cancer-related death worldwide (1) (Siegel et al., 2016). As opposed to the steady increase in survival rates for most cancers, the 5-year survival rates of PC have not improved signi cantly in the past decades and remain less than 5% (2-4)(G and M, 2017; R et al., 2020; Tempero and Margaret, 2017). The dismal prognosis of PC is mainly due to its rapid disease progression and highly metastatic potential (5, 6)(H et al., 2018; Uccello et al., 2018). To date, the diagnosis and treatment of PC relies mostly on imaging methods and curative resection, which are limited and insu cient in many cases (7) (Zhang et al., 2018).
It is reported that most patients with PC are diagnosed at an advanced stage when radical surgery becomes impossible (8,9) (Alatise et al., 2012;Vincent et al., 2011). Therefore, uncovering the molecular mechanism governing the process and metastasis of PC as well as identifying novel diagnostic and therapeutic biomarkers are of utmost clinical importance for patients with PC.
Estrogen-related receptor alpha (ERRα) is an orphan nuclear receptor that structurally similar to estrogen receptors, which could interact with estrogen-responsive elements but not with natural estrogens (10,11) (Felicity, 2014;Welboren et al., 2009). Accumulating evidence indicate that ERRα acts as an oncogenic regulator in the tumorigenesis of a variety of human cancers (12) (Chang and Mcdonnell, 2012). For example, the expression of ERRα was aberrantly higher in gallbladder cancer and correlated with poor prognosis (13) (L et al., 2020). In prostate cancer studies, ERRα has been identi ed as an important negative prognostic factor (14,15) (Fujimura et al., 2007;Z et al., 2020). However, nothing is currently known regarding the expression and role of ERRα in PC.
In the current study, we performed a series of in vitro and in vivo experiments and revealed that, ERRα was overexpressed in PC tissues and negatively correlated with the prognosis of PC patients. ERRα facilitates PC cell proliferation both in vitro and in vivo, and promotes cell migration and invasion via inducing epithelial-mesenchymal transition (EMT) process. In addition, the knockdown of ERRα induces cell apoptosis and cycle arrest in PC cells. Further mechanistic analysis demonstrated that ERRα plays its oncogenic role in PC by enhancing the transcription of plasminogen activator inhibitor 1 (PAI1, also known as SERPINE1) and activating the MEK/ERK signaling pathway. Our study provides the rst data that ERRα might be a novel diagnostic and therapeutic target in PC.

Patient samples and clinicopathologic data
A total of 50 pairs of pancreatic cancer and adjacent non-tumor tissues were obtained from the Department of General Surgery in Xinhua Hospital a liated with Shanghai Jiao Tong University (Shanghai, China) between 2012 and 2016. All cases hadn't received any radiotherapy or chemotherapy before surgery, and were histologically con rmed and staged based on the American Joint Committee on Cancer Staging Manual (8th edition). The informed consent was obtained from all enrolled patients and the study was approved by Ethics Committee of Xinhua Hospital A liated to Shanghai Jiaotong University School of Medicine (Approval No. XHEC-D-2020-076). Immunohistochemistry Immunohistochemistry (IHC) staining was conducted following the standard staining procedure (16)(X et al., 2019). The ERRα expression level was scored based on the percentage of immunoreactive cells: Negative, < 5% immunoreactive cells; Weak, 5%-34% immunoreactive cells; Moderate, 35%-64% immunoreactive cells; Strong, ≥ 65% immunoreactive cells. The total staining score was based on the sum of the extent and intensity, and samples were classi ed as negative (0-1), weak (2-3), moderate (4-5) and strong (6-7) staining. Among all the PC tissues, 34 were identi ed as ERRα-high tissues (moderate and strong staining) and 16 were identi ed as ERRα-low tissues (negative and weak staining) according to the IHC score.

Cell culture and reagents
Pancreatic cancer cell lines (Mia PaCa-2, PaTu8988, PANC1), normal human pancreatic ductal epithelial cell line (HPNE) and 293T cell line were obtained from the Shanghai Key Laboratory of Biliary Tract Disease Research, Shanghai, China. All cell lines were cultured in high-glucose DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco), and incubated in a humidi ed incubator with 5% CO 2 at 37℃.

Cell transfection
ERRα was silenced with small interfering RNAs (siRNAs) using Rfect reagent (BAIDAI) according to the manufacturer's protocol. The sense sequence are as follows: si-ERRα-1 (sense, GCGAGAGGAGUAUGUUCUA; antisense, UAGAACAUACUCCUCUCGC); si-ERRα-2 (sense, GAGAGGAGUAUGUUCUACUAA; antisense, UUAGUAGAACAUACUCCUCUC); si-PAI1 (sense, UCUCUGCCCUCACCAACAUUC; antisense, GAAUGUUGGUGAGGGCAGAGA). The shRNA targeting ERRα was synthesized using the si-ERRα-1 sequence and inserted into PGMLV-SC5 vector, and the full-length sequence of ERRα was cloned into pCDNA3.1 vector (Genomeditech, Shanghai). Empty vectors were used as control. Cells were infected by concentrated lentivirus at a multiplicity of infection (MOI) of 90 for 48 hours. Cell lines were selected by puromycin (1 µg/ml) for 1 week to construct stable transfected cell lines, transfection e ciency was veri ed by qRT-PCR and western blotting. Immuno uorescence assay Cells were xed with 4% paraformaldehyde, and immuno uorescence (IF) assay was conducted using Immunol Fluorence Staining Kit with Cy3 (Beyotime, China) following the manufacturer's instructions. Brie y, cells were permeabilized in 0.1% Triton-X-100, blocked by 3% BSA, incubated with primary antibody at 4℃ overnight and then incubated with Labeled Goat Anti-Rabbit IgG at room temperature away from light. DAPI was used for cell counterstaining. A uorescence microscope (Leica) was used to capture photos.

Western blot analysis
Protein extraction and western blot was performed according to the standard protocol as previously described (17)(S et al., 2019). In brief, equal amounts of proteins were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk, incubated with speci c primary antibodies at 4℃ overnight and then with HRP-conjugated secondary antibody at room temperature for 1 h. Protein signals were detected using a Gel Doc 2000 (Bio-Rad, USA). All primary antibodies were purchased from Cell Signaling Technology.

Cell proliferation assays
Cell Counting Kit-8 (CCK-8) assay and 5-Ethynyl-2′-deoxyuridine (EdU)-488 proliferation assay were performed to evaluate cell proliferation state. For CCK-8 assay, cells were seeded into a 96-well plate at the density of 2000/well with 100 µL complete medium overnight. Each well was then replaced with 10 µL CCK-8 reagent with 90 µL medium, incubated in dark at 37 ℃ for 2 hours and then using SpectraMax 190 Microplate Reader to measure the optical density at 450 nm.
EdU Cell Proliferation Kit (Beyotime) was used to detect cell proliferation according to the manufacturer's instructions. Cells were incubated with 10 µM EdU in complete medium at 37 ℃ for 2 hours. Azide 488 and Hoechst reagent was used for detection of EdU-positive cells and cell counting, respectively. Images were taken under a uorescence microscope (Leica).

Colony formation assay
Transfected cells were plated onto 6-well plates (1000 cells/well) and cultured for 7 days. Then cells were xed with 4% paraformaldehyde for 20 minutes and stained with 0.1% crystal violet stain solution for 15 minutes. Images of the stained plates were photographed and the colonies were counted.

Nude mice xenograft models
Female BALB/c nude mice (4 weeks old, weighted 18-22 g) were purchased from Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. The mice were randomly divided into 4 groups (ERRα, vector, shERRα, vector-shRNA) of 5 mice each and housed under appropriate condition. 2 × 10 6 PaTu8988 cells with stable transfection of LV-ERRα, LV-shERRα or their empty vectors were injected into the left axilla of each mouse. The volume of tumor was measured with calipers weekly using the following formula: 0.5 × width 2 × length. 4 weeks after the cells injection, all mice were sacri ced by cervical dislocation, and the xenograft tumors were carefully dissected and weighed. This animal study was approved by the Ethics Committee of Xinhua Hospital A liated to Shanghai Jiaotong University School of Medicine.

Transwell assays
Transwell migration and invasion assays were applied using chamber inserts (Corning) and BioCoat Matrigel Invasion chamber inserts (Corning), respectively. Treated cells were suspended in 200 µL serumfree medium and seeded in the upper chamber. For both assays, the lower chamber was added with 750 µL complete medium. After 24 hours incubation, cells in the lower surface were xed with 4% paraformaldehyde and stained with 0.1% crystal violet stain solution. 5 random elds of views were photographed under a microscope.
Wound healing assay Cells were seeded in 6-well plates and cultured to more than 90% cell con uence. Wounds were scratched with same strength by use a 200-µL pipette tip. After washing with fresh medium and incubating for 24 hours, images were taken by a microscope at 5 random elds of views. Wound closure percentage was evaluated by comparing the changes before and after 24 hours.

Apoptosis assay
Annexin V-FITC Apoptosis Detection Kit (Beyotime) was used to detect cell apoptotic ratio following the manufacturer's instructions. Transfected cells were harvested and resuspended with binding buffer, and stained with 5 µL Annexin V-FITC and 10 µL propidium iodide (PI). After 20 minutes incubation at room temperature away from light, stained cells were analyzed by ow cytometry (BD Biosciences).

Cell cycle analysis
Cell Cycle Analysis Kit (Beyotime) was used for analysis of cell cycle distribution. Transfected cells were collected and xed with ice-cold 70% ethanol at 4 ℃ overnight. The next day, cells were incubated with RNase A and PI for 30 minutes. DNA content was measured by ow cytometry (BD Biosciences) and analyzed by FlowJo software.

RNA sequencing
Total RNA was extracted from PaTu8988 and PANC1 cells after transfection with si-NC or si-ERRα-1, and then subjected to mRNA sequencing on the BGISEQ-500 platform (Beijing Genomics Institute, China). Of all the differentially expressed genes, 40 most up-regulated genes and 40 most down-regulated genes were selected and analyzed using the BGI online analysis system.

Chromatin immunoprecipitation
The promoter sequence of PAI1 was obtained from NCBI (https://www.ncbi.nlm.nih.gov/), the potential binding sites of ERRα in the promoter region of PAI1 were identi ed by JASPAR Dual-luciferase reporter gene assay 293T cells transfected with full-length ERRα, shERRα or their empty vectors were seeded onto 24-well plates and co-transfected with indicated luciferase reporters. pGL3-PAI1-WT-luc (containing the − 2000 bp to -1 bp promoter sequence of PAI1) and pGL3-PAI1-MUT-luc (the 2 binding sites of ERRα in the promoter region of PAI1 were mutated) were synthesized by Genomeditech. At 48 hours post-transfection, luciferase activities were determined by a Dual-Luciferase Reporter Assay System (Promega) following the manufacturer's instructions. The relative luciferase activities were presented after normalization to Renilla luciferase activity.

Statistical analysis
All experiments were repeated at least three times. Data were analyzed using Prism 8 software and presented as the mean ± standard deviations (SD). The Student's test was performed for the comparisons between two groups, and Pearson chi-square test was used to analyze the correlation between ERRα expression and clinicopathologic variables. Survival analysis was applied by Kaplan-Meier method and log-rank test. P < 0.05 was considered statistically signi cant.

Results
Up-regulated expression of ERRα is associated with poor prognosis in human PC To identify the biological role of ERRα in PC, we rst performed qRT-PCR to detect the mRNA levels of ERRα in 50 pairs of PC tissues and adjacent non-tumor tissues. As shown in Fig. 1A-B, ERRα mRNA expression was signi cantly higher in PC tissues than in adjacent non-tumor tissues. We also conducted IHC assay to compare the protein expression of ERRα in these samples (Fig. 1D). The results of IHC were consistent with that of qRT-PCR to suggest an up-regulation of ERRα in PC tissues at the protein level (Fig. 1E). Then we determined the relationship between ERRα and clinical features in PC patients. As shown in Table 1, high ERRα expression was associated with tumor size (P = 0.0065), distant metastasis (P = 0.0026), TNM stage (P = 0.0021) and tumor differentiation (P = 0.0045). Moreover, Kaplan-Meier analysis of overall survival (OS) revealed that upregulated expression of ERRα was correlated with poor prognosis in PC (P < 0.0001, Fig. 1C). ERRα expression in PC cell lines We performed IF assay, qRT-PCR and western blot assay to detect the expression of ERRα in PC cell lines (PaTu8988, PANC1 and Mia PaCa-2) and HPNE. The results revealed that ERRα was expressed in the nuclei of cells and overexpressed in PC cell lines compared with HPNE ( Fig. 2A-C). To investigate further, we knocked down ERRα in PaTu8988 and PANC1 cells using siRNAs and overexpressed ERRα in Mia PaCa-2 cells. The transfection e ciency was validated by qRT-PCR and western blot analysis (Fig. 2D).
ERRα promotes PC proliferation in vitro and in vivo.
To explore the effects of ERRα on PC cell proliferation in vitro, we conducted CCK-8 assay, EdU-488 proliferation assay and colony formation assay. As shown in Fig. 3A-C, the proliferation ability of PC cells was markedly suppressed after ERRα depletion, whereas was enhanced after ERRα overexpression. We also investigated the effect of ERRα on PC tumor growth in vivo. We used PaTu8988 cells with stable transfection of LV-ERRα, LV-shERRα or their empty vectors (Fig. 3D) to construct xenograft models. As indicated by the volume, weight and growth curves of the tumors, ERRα overexpression signi cantly promoted the growth of PaTu8988 cells in xenograft models, whereas ERRα silencing did the opposite ( Fig. 3E and F). These results indicated a growth-promoting role for ERRα on PC in vitro and in vivo.

ERRα facilitates PC cell migration and invasion through EMT
To determine the effect of ERRα on PC metastasis, we assessed PC cell migration and invasion abilities in vitro using wound healing assay as well as transwell migration and invasion assays. As shown in Fig. 4A-D, ERRα depletion notably suppressed the migration and invasion ability of PaTu8988 and PANC1 cells compared with negative controls. Conversely, ERRα overexpression remarkably enhanced the cell migration and invasion in Mia PaCa-2 cells. As the EMT process played a vital role in the cancer metastasis (18)(I and C, 2019), we explored whether EMT was involved in the effect of ERRα on migration and invasion. As shown in Fig. 4E and F, knockdown of ERRα signi cantly increased the expression levels of epithelial marker E-cadherin, but decreased the expression levels of mesenchymal markers N-cadherin, Vimentin and Snail. What's more, the overexpression of ERRα presented exactly the opposite results. Collectively, these results indicated that ERRα promotes the ability of migration and invasion via inducing EMT in PC cells.

Knockdown of ERRα induces cell apoptosis and cycle arrest in PC cells
To assess whether the proliferation defects observed in ERRα-depleted PC cells were due to the cell apoptosis and arrest of cell cycle progression, we performed ow cytometry assays to evaluate the effects of ERRα knockdown on PC cell apoptosis and cell cycle regulation. The results demonstrated that ERRα silencing signi cantly increased the apoptosis rate in PC cells, and the analysis of cell cycle distribution showed that the proportion of G0/G1 phase cells was remarkably increased and that of Sphase cells was decreased after ERRα knockdown ( Fig. 5A and C). In addition, we detected the expression of apoptosis-related and cell cycle-related markers by western blotting and IF assay (Fig. 5B, D and E). The data revealed that protein expression levels of cleaved-PARP, cleaved-caspase 3, cytochrome c, p21 Waf1/Cip1 and p27 Kip1 were signi cantly increased while that of bcl-2, CDK2, CDK4 and cyclin D1 were decreased after ERRα knockdown. Moreover, ERRα overexpression led to diametrically opposite results. Taken together, the above results provide more insight into the mechanism and indicate that knockdown of ERRα inhibits PC cell proliferation by inducing apoptosis and arrest of the G0/G1 phase.
PAI is upregulated and correlated with poor prognosis in PC RNA-sequencing was performed to further explore the potential mechanism by which ERRα affects pancreatic cancer progression. Compared with the negative control cells, we selected the top 40 downregulated and top 40 up-regulated genes in PaTu8988 and PANC1 cells after ERRα knockdown, among which the plasminogen activator inhibitor 1 (fold change= -1.600, p < 0.001) attracted our attention (Fig. 6A). PAI1, a serine protease inhibitor, has been reported to be involved in the development and  Fig. 6D). Then the expression levels of PAI1 in PC cell lines and HPNE cell line were determined by qRT-PCR and western blotting. The results demonstrated that PAI1 was upregulated in PC cell lines compared with HPNE cells (Fig. 6E). In addition, the expression levels of PAI1 could be decreased by transfection with si-ERRα-1 or si-PAI1 and increased by ERRα overexpression (Fig. 6F).

PAI1 mediates the oncogenic behavior of ERRα in PC cells.
We then investigated whether PAI1 is involved in ERRα-mediated oncogenic effects on PC cells. As shown in Fig. 7A-E, the knockdown of PAI1 remarkably inhibited the cell proliferation, migration and invasion in PaTu8988 and PANC1 cells. More importantly, PAI1 depletion could rescue the cell proliferation, migration and invasion enhanced by ERRα overexpression in Mia PaCa-2 cells. Therefore, it is hypothesized that PAI1 participates in the ERRα-induced oncogenic effects in PC.
ERRα enhances the transcription of PAI1 via directly binding to its promoter We then investigated the mechanisms responsible for ERRα regulated PAI1. Bioinformatic analysis was performed using JASPAR database and 2 high-scoring predicted binding sites of ERRα in the PAI1 promoter sequence were found (Fig. 8A and B). ChIP assays were conducted in PaTu8988 cells to con rm the bounding relationship between ERRα protein and PAI1 promoter. ChIP samples were further analyzed via DNA electrophoresis and qPCR. The results of DNA electrophoresis showed the predicted DNA band in Input and ERRα groups but not in the IgG group (Fig. 8C), and the results of qPCR con rmed the enrichment of PAI1 promoter in ERRα precipitates (Fig. 8D).
Subsequent dual luciferase assays demonstrated that the luciferase activity of WT PAI1 promoter reporter was notably increased by ERRα overexpression and decreased by ERRα inhibition. What's more, the luciferase activity of MUT PAI1 promoter was unaffected by ERRα (Fig. 8E). Together, these data indicated that ERRα could bind to the promoter region of PAI1 and transcriptionally enhance the expression of PAI1.

ERRα promotes PC progression through the MEK/ERK signaling pathway
Previous studies have reported that ERRα is associated with the PI3K/AKT pathway (22,23) ERRα overexpression (Fig. 8G and H). These results indicate that ERRα promotes PC progression via activating the MEK/ERK signaling pathway.

Discussion
Accumulating evidence has con rmed the aberrant expression of ERRα in human cancers, and increasing attention has been focused on the vital role of ERRα in the processes related to cancer progression, such as tumor growth, metastasis and chemoresistance (14,16,26,27) patients. Subsequently, a series of in vitro and in vivo assays were conducted and the results showed that, ERRα promoted PC cell proliferation, migration, invasion as well as induced cell apoptosis and G0/G1cycle arrest via enhancing the transcription of PAI1 and activating the MEK/ERK signaling pathway. To our knowledge, this is the rst study to identify the critical role of ERRα in PC progression, suggesting that ERRα may be a novel diagnostic and therapeutic target for PC.
The present study revealed that ERRα was highly expressed in PC cell lines and knockdown of ERRα could suppress the proliferation and colony formation of PC cells. In addition, EdU proliferation assays demonstrated that ERRα inhibition notably reduced the percentage of EdU-positive cells, suggesting a decreased in DNA replication. Consistent with the in vitro assays, our in vivo tumor growth assay showed that ERRα overexpression increased the PC growth whereas ERRα silencing exerted the opposite effect. Furthermore, ow cytometry analysis indicated that the suppressive effects of ERRα knockdown on the proliferation of PC may be due to induction of cell apoptosis and cell cycle arrest. Apoptosis is a crucial gene-directed program that is engaged to regulate normal cellular growth and development (28)    The expression of ERRα in PC cell lines. A, Immuno uorescence staining of ERRα (red) in PC cell lines and normal human pancreatic ductal epithelial cell line (HPNE). Nucleus was stained by DAPI (blue). B and C, The mRNA and protein expression levels of ERRα in PC cell lines and HPNE. D, Knockdown of ERRα in PaTu8988 and PANC1 cells, and overexpression of ERRα in Mia PaCa-2 cells was con rmed at the mRNA and protein level by qRT-PCR and western blotting. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3
ERRα promotes PC cell proliferation and tumor growth. A-C, CCK-8, EdU and colony formation assays were applied to determine the proliferation of PaTu8988 and PANC1 cells after ERRα depletion, and that of Mia PaCa-2 cells after ERRα overexpression. D, The mRNA and protein expression level of ERRα in PaTu8988 cells transfected with LV-ERRα, LV-shERRα or their empty vectors. E, Compared with empty vector groups, overexpression of ERRα promoted while knockdown of ERRα suppressed the tumor growth in nude mice. F, Tumor weight was measured after mice sacri ced and tumor resection; Tumor growth curves were plotted. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5
Knockdown of ERRα induces cell apoptosis and G0/G1 arrest in PC cells. A, Flow cytometric analysis of apoptosis in PaTu8988 and PANC1 cell after ERRα knockdown. B, Western blot analysis of apoptosis related markers. C, After silencing of ERRα, ow cytometry showed that the cell cycles of PaTu8988 and PANC1 cells were arrested at G0/G1 phase. D, Cell cycle related proteins were analyzed by western blotting. E, The expression levels of cleaved-PARP, cleaved-caspase 3, cyclin D1 and p27 Kip1 were detected by IF assay. *p < 0.05, **p < 0.01, ***p < 0.001.   ERRα promotes the transcription of PAI1 and activates the MEK/ERK pathway. A and B, ERRα binding motif and the predicted ERRα binding sites within the promoter region of PAI1 according to the JASPAR database. C and D, ChIP-DNA electrophoresis and ChIP-qPCR using primers designed for the 2 predicted sites validated the binding capacity of ERRα to the PAI1 promoter. E, Dual-luciferase reporter assay showed that ERRα overexpression signi cantly increased while ERRα knockdown suppressed the activity of the wild-type PAI1 promoter luciferase reporter. Moreover, ERRα had no effect on the activity of mutant PAI1 promoter luciferase reporter. F, The expression levels of p-MEK1/2 and p-ERK1/2 were suppressed by ERRα knockdown; ERRα overexpression enhanced the expression of p-MEK1/2 and p-ERK1/2, which was abolished by knockdown of PAI1. G and H, The cell proliferation, migration and invasion promoted by ERRα overexpression was signi cantly antagonized by ERK1/2 inhibitor GDC-0994.