Notch-1 induces epithelial-mesenchymal transition consistent with cancer stem cell phenotype in pancreatic cancer cells

Abstract

Activation of Notch-1 is known to be associated with the development and progression of human malignancies including pancreatic cancer. Emerging evidence suggest that the acquisition of epithelial-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotype are interrelated and contributes to tumor recurrence and drug resistance. The molecular mechanism(s) by which Notch-1 contributes to the acquisition of EMT phenotype and CSC self-renewal capacity has not been fully elucidated. Here we show that forced over-expression of Notch-1 leads to increased cell growth, clonogenicity, migration and invasion of AsPC-1 cells. Moreover, over-expression of Notch-1 led to the induction of EMT phenotype by activation of mesenchymal cell markers such as ZEB1, CD44, EpCAM, and Hes-1. Here we also report, for the first time, that over-expression of Notch-1 leads to increased expression of miR-21, and decreased expression of miR-200b, miR-200c, let-7a, let-7b, and let-7c. Re-expression of miR-200b led to decreased expression of ZEB1, and vimentin, and increased expression of E-cadherin. Over-expression of Notch-1 also increased the formation of pancreatospheres consistent with expression of CSC surface markers CD44 and EpCAM. Finally, we found that genistein, a known natural anti-tumor agent inhibited cell growth, clonogenicity, migration, invasion, EMT phenotype, formation of pancreatospheres and expression of CD44 and EpCAM. These results suggest that the activation of Notch-1 signaling contributes to the acquisition of EMT phenotype, which is in part mediated through the regulation of miR-200b and CSC self-renewal capacity, and these processes could be attenuated by genistein treatment.

Figure 1

Over-expression of Notch-1 increased cell growth (A), the relative protein levels of cyclin D1, VEGF, p65, Hes-1, ZEB1, CD44, and EpCAM (B), the acquisition of EMT phenotype (C), over-expression of Notch-1 differentially regulated the miRNA expression of let7a, b, c, miR-21, and miR-200b, c (D) in human pancreatic cancer AsPC-1 cells. Notch-1 over-expressing AsPC-1 cells were established by stable gene transfection technique. The relative levels of miRNAs were measured by real-time RT-PCR technique

Figure 2

Re-expression of miR-200b regulated the expression of EMT phenotype marker mRNAs (A) and proteins (B), and reversed EMT phenotype (C) in Notch-1 over-expressing AsPC-1 cells. Re-expression of miR-200b was established in Notch-1 over-expressing AsPC-1 cells by transfection with its precursor. Real-time RT-PCR and Western blotting analysis were conducted to measure the relative levels of mRNAs and proteins, respectively.

Figure 3

Genistein treatment decreased cell survival (A), clonogenicity (B), relative levels of proteins (C) in AsPC-1-control and AsPC-1-Notch-1 cells. Different concentrations of genistein were used and the cells were exposed for 3 days. The cells were then harvested for MTT assay, clonogenic assay, and Western blotting analysis, respectively.

Figure 4

Genistein treatment decreased cell migration and invasion in AsPC-1-control and AsPC-1-Notch-1 cells. Wound healing assay was conducted to assess the capacity of cell migration and invasion in AsPC-1 cells.

Figure 5

Genistein treatment decreased the capacity of CSC-self-renewal in primary (A) and secondary (B, C) pancreatospheres of AsPC-1-control and AsPC-1-Notch-1cells. The sphere forming assay was conducted in AsPC-1-control and AsPC-1-Notch-1cells in the absence or presence of genistein for 1 or 3 weeks of treatment

Figure 6

Genistein treatment decreased the protein expression of CSC surface markers, CD44 and EpCAM in pancreatospheres of AsPC-1-control and AsPC-1-Notch-1cells. Immunostaining and confocal microscopy (Magnification X 250) were conducted in pancreatospheres of AsPC-1-control and AsPC-1-Notch-1cells after 1 week of genistein treatment.