Ezrin promotes invasion and metastasis of pancreatic cancer cells

Abstract

Background: Pancreatic cancer has a high mortality rate because it is usually diagnosed when metastasis have already occurred (microscopic and gross disease). Ezrin plays important roles in cell motility, invasion and tumor progression, and it is especially crucial for metastasis. However, its function in pancreatic cancer remains elusive.

Methods and results: We found that ezrin overexpression promoted cell protrusion, microvillus formation, anchorage-independent growth, motility and invasion in a pancreatic cancer cell line, MiaPaCa-2, whereas ezrin silencing resulted in the opposite effects. Ezrin overexpression also increased the number of metastatic foci (6/8 vs. 1/8) in a spontaneous metastasis nude mouse model. Furthermore, ezrin overexpression activated Erk1/2 in MiaPaCa-2 cells, which might be partially related to the alteration of cell morphology and invasion. Immunohistochemical analysis showed that ezrin was overexpressed in pancreatic ductal adenocarcinoma (PDAC) (91.4%) and precancerous lesions, i.e. the tubular complexes in chronic pancreatitis (CP) and pancreatic intraepithelial neoplasm (PanIN) (85.7% and 97.1%, respectively), compared to normal pancreatic tissues (0%). Ezrin was also expressed in intercalated ducts adjacent to the adenocarcinoma, which has been considered to be the origin of ducts and acini, as well as the starting point of pancreatic ductal carcinoma development.

Conclusions: We propose that ezrin might play functional roles in modulating morphology, growth, motility and invasion of pancreatic cancer cells, and that the Erk1/2 pathway may be involved in these roles. Moreover, ezrin may participate in the early events of PDAC development and may promote its progression to the advanced stage.

Figure 1

Stable overexpression and silencing of ezrin in MiaPaCa-2 cells. (A) Western blot showed the ezrin protein was overexpressed in the Mia ez22-B cells compared to the Mia pcb6 cells using an ezrin antibody. The relative ezrin protein level was quantified by densitometry analysis. The ezrin protein was efficiently increased by 3.8 folds in the Mia ez22-B cells. (B) Ectopic expression of ezrin in the Mia ez22-B cells was detected using a VSV-G antibody (VSV-G tag in the pcb6-ezrin vector). (C) The expression level of ezrin protein was dramatically decreased by 70.5% and 90.1% in the Mia ezsi-B and the Mia ezsi-E cells, respectively, compared to that in the Mia ezsi-scram cells. GAPDH was used as a loading control. (D) The Mia pcb6 cells were stained with the ezrin antibody and a FITC-conjugated second antibody to detect the ezrin protein expression. (E) The vector tag VSV-G antibody and a Rhodamine-conjugated second antibody were used to detect the exogenous ezrin protein expression. (F, G) The Mia ezsi-scram and the Mia ezsi-E cells were stained with the ezrin antibody and the FITC-conjugated second antibody to detect the ezrin protein expression.

Figure 2

Scanning electron microscopy showed increased formation of membrane protrusions and microvilli in the Mia ez22-B cells (B) compared to that in the Mia pcb6 cells (A). A sharp decrease of the membrane protrusions and smooth edge in the Mia ezsi-E cells (D) compared to those in the Mia ezsi-scram cells (C).

Figure 3

Effects of ezrin on MiaPaCa-2 cell growth and anchorage-independent growth. (A) The cell growth curves of the Mia ezsi-scram, Mia ezsi-E, Mia pcb6 and Mia ez22-B cells were assayed on days 1-7. (B) Flow cytometry assay showing the percentage of different cell cycle phases in the four cell clones. (C) Anchorage-independent growth assay of ezrin-overexpressing and ezrin-silencing cells. The cell growth ability in soft agar of the four cell clones was examined for three weeks. Columns, mean; bars, SD. (D) Statistical analysis of colony formation in the four cell clones. There was a significant difference of the colony formation ability between the Mia ez22-B and the Mia pcb6 cells, as well as between the Mia ezsi-scram and the Mia ezsi-E cells, respectively, shown by x²-test. The results are expressed as the mean ± SD of three independent experiments.

Figure 4

Effects of ezrin on cell motility in vitro. BioCoat Chambers were used to detect cell migration and representative fields were photographed. Black-arrows indicate the 8-µm membrane pores, and hollow-arrows indicate cells that had migrated through the membrane, which were stained with Crystal Violet (a). Cell migration of the Mia ez22 (b), Mia pcb6 (a), Mia ezsi-E (e) and Mia ezsi-cram (d) cells after 12 hours were shown. The cells migrating to the lower chambers were analyzed. For quantification, the cells were counted in 10 random fields under a light microscope (×400). Compared to the Mia pcb6 cells, the Mia ez22-B cells showed a significant increase in migration by x²-test (c). The decrease in the numbers of migrated cells in the Mia ezsi-E cells compared to those of the Mia ezsi-scram cells was statistically significant, shown by the x²-test (f). Columns: mean; bars: SD.

Figure 5

Effects of ezrin on cell invasion in vitro. Matrigel-coated transwell chambers were used to detect cell invasion and representative fields were photographed. Cell invasion of the Mia ez22 (b), Mia pcb6 (a), Mia ezsi-E (e) and Mia ezsi-cram (d) cells after 24 hours were shown. The cells invading to the lower chambers were analyzed. Compared to the Mia pcb6 cells, the Mia ez22-B cells showed a significant increase in invasion by x²-test (c). The decrease in the numbers of invasive cells in the Mia ezsi-E cells compared to those of the Mia ezsi-scram cells was statistically significant, shown by the x²-test (f). Columns: mean; bars: SD.

Figure 6

Ezrin overexpression increasing the level of phosphorylated Erk1/2 in MiaPaCa-2 cells. The levels of phosphorylated-ezrin, total AKT, phosphorylated-AKT, total Erk1/2 and phosphorylated Erk1/2 were determined by western blot in the Mia ezsi-scram, Mia ezsi-E, Mia pcb6 and Mia ez22-B cells. GAPDH was used as a loading control.

Figure 7

Ezrin expression in normal pancreatic tissue and pancreatic ductal adenocarcinoma shown by immunohistochemistry. (A) Normal pancreatic tissue. (B) Well-differentiated pancreatic ductal adenocarcinoma. (C) Moderate-differentiated pancreatic ductal adenocarcinoma. (D) Poor-differentiated pancreatic ductal adenocarcinoma.

Figure 8

Expression of ezrin in intercalated duct cells, the tubular complexes of CP and PanINs. (A) Staining of ezrin in the intercalated duct cells in the paraneoplastic tissues. (B-D) Ezrin-positive staining in the representative clinical specimens of PanIN1, PanIN2 and PanIN3 (×100 magnification). (E) Ezrin expression in the cells of the tubular complexes in CP (×100 magnification).