Yin Yang-1 suppresses invasion and metastasis of pancreatic ductal adenocarcinoma by downregulating MMP10 in a MUC4/ErbB2/p38/MEF2C-dependent mechanism

Abstract

Background: Increasing evidence indicates an important role of transcription factor Yin Yang-1 (YY1) in human tumorigenesis. However, its function in cancer remains controversial and the relevance of YY1 to pancreatic ductal adenocarcinoma (PDAC) remains to be clarified.

Methods: In this study, we detected YY1 expression in clinical PDAC tissue samples and cell lines using quantitative RT-PCR, immunohistochemistry and western blotting. We also detected MUC4 and MMP10 mRNA levels in 108 PDAC samples using qRT-PCR and analyzed the correlations between YY1 and MUC4 or MMP10 expression. The role of YY1 in the proliferation, invasion and metastatic abilities of PDAC cells in vitro was studied by CCK-8 assay, cell migration and invasion assays. In vivo pancreatic tumor growth and metastasis was studied by a xenogenous subcutaneously implant model and a tail vein metastasis model. The potential mechanisms underlying YY1 mediated tumor progression in PDAC were explored by digital gene expression (DGE) sequencing, signal transduction pathways blockage experiments and luciferase assays. Statistical analysis was performed using the SPSS 15.0 software.

Results: We found that the expression of YY1 in PDACs was higher compared with their adjacent non-tumorous tissues and normal pancreas tissues. However, PDAC patients with high level overexpression of YY1 had better outcome than those with low level overexpression. YY1 expression levels were statistically negatively correlated with MMP10 expression levels, but not correlated with MUC4 expression levels. YY1 overexpression suppressed, whereas YY1 knockdown enhanced, the proliferation, invasion and metastatic properties of BXPC-3 cells, both in vitro and in vivo. YY1 suppresses invasion and metastasis of pancreatic cancer cells by downregulating MMP10 in a MUC4/ErbB2/p38/MEF2C-dependent mechanism.

Conclusions: The present study suggested that YY1 plays a negative role, i.e. is a tumor suppressor, in PDAC, and may become a valuable diagnostic and prognostic marker of PDAC.

Figure 1

YY1 expression in PDAC tissues and cell lines. (a, b) qRT-PCR analysis of YY1 expression in five normal pancreas tissues and 108 pairs of PDAC tissue and adjacent tissue. YY1 expression in the normal pancreas tissues was low, but was slightly higher in the tissue adjacent to PDAC tumors and significantly higher in the PDAC tumors themselves. Of the 108 paired tissues, YY1 expression was higher in the cancer tissue than in the adjacent tissues in 86 cases (p < 0.05). (c) A boxplot showing the qRT-PCR results of YY1 mRNA expression in the 108 cancer tissue, 108 adjacent tissue and five normal pancreas tissues (mean ± SD: 1.01 ± 1.01, 0.38 ± 0.43 and 0.03 ± 0.01, respectively). The bottom and the top edges of the box mark the lower boundary and upper boundary of the 95% confidence interval (CI) for the mean. The center horizontal line is drawn at the sample mean. The center vertical lines drawn from the boxes extend to the minimum and the maximum. (d) qRT-PCR analysis of YY1 expression in six PDAC cell lines. (e) Western blotting of YY1 expression in six PDAC cell lines. (f) A boxplot showing the immunohistochemistry (IHC) results of YY1 protein expression in the 108 cancer tissue, 108 adjacent tissue and five normal pancreas tissues. (g) IHC showing the location of YY1 in PDAC samples. Scale bar, 100 μm. (** represents p < 0.01, # represents p < 0.001, compared with the control group).

Figure 2

Correlation between YY1 expression and patient survival. (a) ROC curve for YY1 expression and cut-off value selection for high and low level YY1 expression. (b) Kaplan-Meier survival curves in 108 patients with PDAC according to their YY1 mRNA expression status. The p value was calculated by the Log-rank test.

Figure 3

Effects of YY1 on the proliferation, migration and invasion of BXPC-3 cells in vitro. (a) Measurement of YY1 expression in stably transfected BXPC-3 cells using qRT-PCR. BXPC-YY1 indicates YY1overexpressing BXPC-3 cells; BXPC-Vector indicates BXPC-3 cells transfected with a control vector; BXPC-YY1 shRNA indicates YY1 knockdown BXPC-3 cells; BXPC-Scramble shRNA indicates BXPC-3 cells transfected with a vector expressing Scramble shRNA. (b) Measurement of YY1 expression in stably transfected BXPC-3 cells using western blotting. The results are similar to those seen in the qRT-PCR analysis. (c) CCK-8 assays were performed. OD values from four independent experiments were assessed. (d) Cell migration and invasion assays were performed. The upper chambers were seeded with various cell lines. The membranes of the chambers were stained with 0.1% crystal violet. Scale bar, 100 μm. (e, f) Quantification of the data from Figure 3d. OD values from three independent experiments were assessed. (*represents p < 0.05, **represents p < 0.01, # represents p < 0.001, compared with the control group).

Figure 4

Effects of YY1 on the tumor growth and metastasis of BXPC-3 cells in vivo. (a) BXPC-Scramble shRNA and BXPC-YY1 shRNA cells were bilaterally injected into the flank region of the mice (1.5 × 10⁶ cells/100 μl per flank). After injection, the length and width of the tumors were measured every week. The volume of the tumors was calculated using the formula (width² × length)/2. The data are presented as means ± SD of 16 tumors for each group. (b) HE staining of the subcutaneous tumors excised four weeks later. Scale bar, 100 μm. (c) Three tumors from each group were resected, RNA was extracted and YY1 expression was measured using qRT-PCR. (d) BXPC-Vector and BXPC-YY1 cells (1.5 × 10⁶ cells/100 μl) were separately injected into the tail vein of each mouse. Four weeks later, lung and liver metastases were evaluated by macroscopic observation and by histomorphology under microscopy. Scale bar, 100 μm. The arrows indicate the metastases. (* represents p < 0.05, # represents p < 0.001, compared with the control group).

Figure 5

Mechanisms involved in the tumor suppression role of YY1. (a) Effects of YY1 on MUC4, ErbB2, MEF2C, MMP10 and MAPK (Erk1/2, JNK and p38) signaling pathways were assayed by western blotting. BXPC-YY1 indicates YY1overexpressing BXPC-3 cells; BXPC-Vector indicates BXPC-3 cells transfected with a control vector; BXPC-YY1 shRNA indicates YY1 knockdown BXPC-3 cells; BXPC-Scramble shRNA indicates BXPC-3 cells transfected with a vector expressing Scramble shRNA. GAPDH was used as the internal control. (b) Bands were quantified by densitometry. Histograms of the ratio (phosphorylated/constitutive form) for Erk1/2, JNK and p38 MAPK kinases are shown. (c) BXPC-YY1 shRNA cells were transfected with MMP10 siRNA or negative control siRNA. 12 h after transfection, cell migration assays were performed. The upper chambers were seeded with various cell lines. The membranes of the chambers were stained with 0.1% crystal violet. Scale bar, 100 μm. (d) Quantification of the data from Figure 5c. OD values from three independent experiments were assessed. (e) Luciferase activities of MMP10 promoter-transfected BXPC-Scramble shRNA or BXPC-YY1 shRNA cells treated with MUC4 siRNA or various inhibitors of signal transduction molecules. MUC4 blockage, the ErbB2 inhibitor (AG825, 25 μM) and the p38 inhibitor (SB203580, 10 μM) significantly inhibited YY1 knockdown-stimulated luciferase activity in BXPC-YY1 shRNA cells transfected with the MMP10 promoter. (f) The YY1 knockdown-stimulated luciferase activity could be significantly inhibited by MEF2C blockage. Luciferase activity of MMP10 promoter in BXPC-YY1 shRNA cells was significantly decreased when the presumed MEF2C binding site (nucleotides −881 to −890) was mutated. (*represents p < 0.05, **represents p < 0.01, # represents p < 0.001, when compared to the control group).

Figure 6

Schematic representation of the roles of YY1 on the migrating and invasive properties of BXPC-3 cells. YY1 suppresses invasion and metastasis of BXPC-3 cells by downregulating MMP10 in a MUC4/ErbB2/p38/MEF2C-dependent mechanism. “?” represents other undiscovered mechanisms that might be involved.