Effect of NR5A2 inhibition on pancreatic cancer stem cell (CSC) properties and epithelial-mesenchymal transition (EMT) markers

Abstract

NR5A2 (aka LRH-1) has been identified as a pancreatic cancer susceptibility gene with missing biological link. This study aims to demonstrate expression and potential role of NR5A2 in pancreatic cancer. NR5A2 expression was quantified in resected pancreatic ductal adenocarcinomas and the normal adjacent tissues of 134 patients by immunohistochemistry. The intensity and extent of NR5A2 staining was quantified and analyzed in association with overall survival (OS). The impact of NR5A2 knockdown on pancreatic cancer stem cell (CSC) properties and epithelial-mesenchymal transition (EMT) markers was examined in cancer cells using RT-PCR and Western Blot. NR5A2 was overexpressed in pancreatic tumors, the IHC-staining H score (mean ± SE) was 96.4 ± 8.3 in normal versus 137.9 ± 8.2 in tumor tissues (P < 0.0001). Patients with a higher NR5A2 expression had a median survival time 18.4 months compared to 23.7 months for those with low IHC H scores (P = 0.019). The hazard ratio of death (95% confidence interval) was 1.60 (1.07-2.41) after adjusting for disease stage and tumor grade (P = 0.023). NR5A2 was highly expressed in pancreatic cancer sphere forming cells. NR5A2-inhibition by siRNA was associated with reduced sphere formation and decreased levels of CSCs markers NANOG, OCT4, LIN28B, and NOTCH1. NR5A2 knockdown also resulted in reduced expression of FGB, MMP2, MMP3, MMMP9, SNAIL, and TWIST, increased expression of epithelial markers E-cadherin and β-catenin, and a lower expression of mesenchymal marker Vimentin. Taken together, our findings suggest that NR5A2 could play a role in CSC stemness and EMT in pancreatic cancer, which may contribute to the worse clinical outcome.

Keywords: EMT; expression; stem cell-like cancer cell; survival.

Figure 1

NR5A2 mRNA expression in human pancreatic ductal epithelial (HPDE) and carcinoma cell lines. Expression of mRNA was measured by RT-PCR (top panel) and protein by Western blot (lower panel). For Western blot the samples were treated with primary antibody (1:1000 dilution, Santa Cruz), imaged by enhanced chemiluminescence, and the results showed normalized expression of NR5A2 protein in the corresponding cells. GAPDH and β-actin was used as internal control for RT-PCR and Western blot, respectively. The mRNA and protein expression levels were consistent and the highest level was seen in AsPC-1 and PANC-1 cells.

Figure 2

Representative micrographs of NR5A2 in pancreatic adenocarcinoma cancerous tissue or its surrounding non-tumor tissue by immunohistochemistry. Images (A and B) show light cytoplasmic and nuclear expression of NR5A2 in normal pancreatic tissues. Images (C and D) show high level of cytoplasmic expression of NR5A2 in pancreatic ductal adenocarcinoma tumor tissues. Magnifications: 40× for A and C; 200× for B and D. (E) Patients with a higher NR5A2 expression in the tumors (n=68) had a significantly reduced OS compared to those with a lower level of expression (n=66). Using the median value of the total IHC score as the cutoff the median survival time was 19.4 vs 23.7 months for those with a high (solid line) or low (dashed line) IHC score, respectively (P=0.021).

Figure 3

Characterization of CSCs in pancreatic cancer cell lines. (A) Microscopy images of adherent cells and corresponding spheres in PANC-1 and AsPC-1 cells, Magnifications: 100× (B) Fold difference of mRNA expression levels of stem cell markers in sphere as compared to that of adherent PANC-1 and AsPC-1 cells. (C) Western blots of adherent cells and corresponding spheres in PANC-1 and AsPC-1 cells. (D) Densitometric analysis showed increased expression of CD44, EPCAM, OCT4 and NANOG proteins in PANC-1 and AsPC-1 spheres as compared to that in the corresponding adherent cells. The expression levels of proteins are normalized to those of β-actin. (E) Flow cytometry analysis for CD24, CD44, c-MET and ALDH expression in AsPC-1 adherent (upper panels) and sphere-forming cells (lower panels). (F) CD24 was expressed in 51.0 ± 0.03% (mean ± SD) of adherent and 92.5 ± 9.9% sphere-forming AsPC-1 cells. Similarly, CD44 was expressed in 44.1 ± 5.4% vs 94.8 ± 6.6%, c-MET in 37.4 ± 7.2% vs 93.2 ± 9.6%, and ALDH in 43.3 ± 8.4% vs 91.9 ± 11.8% of the adherent and sphere-forming cells, respectively.

Figure 4

Blocking NR5A2 by specific siRNA in pancreatic CSCs. Adherent cells of AsPC-1 and PANC-1 were trypsinized and suspended in DMEM/F12 growth medium supplemented with B27 and N2. siPORT™ NeoFX™ Transfection Agent with 10 nM negative siRNA control (si control) or 10 nM anti-NR5A2 siRNAs (siRNA1 or siRNA2) were complexed and dispensed into ultra-low adherent six-well plate, then cell suspensions overlaid onto the transfection complexes and gently tilt the plate to mix. The final density of suspension cells were 10⁴ cells/mL. (A) Microscopy images showing spheres 7 days after NR5A2 knockdown in AsPC-1 and PANC-1 CSCs. Magnifications: 40×. (B) Quantitative RT–PCR showing NR5A2 mRNA expression in spheres transfected with negative control siRNA (si control) and anti-NR5A2 siRNAs (siRNA1 and siRNA2) at 6 days following the transfections. (C) Western blots showing NR5A2 protein expression in spheres transfected with control and anti- NR5A2 siRNAs. Data are shown at 9 days after transfection. Expression of β-actin was used as an internal control.

Figure 5

Gene expression profiling of stem cell markers in pancreatic CSCs. Panels A and B: reduced mRNA expression of stem cell marker genes in NR5A2-inhibited PANC-1 and AsPC-1 CSCs as determined by RT-PCR. Relative mRNA levels were normalized to GAPDH. Panels C and D: significantly decreased expressions of OCT4, NOTCH, NANOG and EZH2 proteins in NR5A2-inhibited PANC-1 and AsPC-1 CSCs. Expression of β-actin was used as an internal control.

Figure 6

NR5A2 regulates EMT-related marker genes in pancreatic CSCs. Panel A and B showed decreased mRNA expression of EMT-related transcription factor such as FGB, FN1, MMP2, MMP3, MMP9, TWIST, SNAIL, and ZEB1 and the mesenchymal marker Vimentin, and increased expression of the epithelial marker E-cadherin in NR5A2-inhibited PANC-1 and AsPC-1 CSCs compared with controls. Relative mRNA levels were normalized to GAPDH. Panel C and D showed reduced protein expression of the mesenchymal marker Vimentin and increased expression of the epithelial marker E-cadherin and β-catenin in NR5A2-inhibited PANC-1 and AsPC-1 CSCs compared with controls. Expression of β-actin was used as an internal control. Panel E showed the increased expression of membrane β-catenin in NR5A2-inhibited AsPC-1 sphere-forming cells compared to that in control siRNA-transfected cells by flow cytometry.