Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells

Abstract

FoxM1 is known to play important role in the development and progression of many malignancies including pancreatic cancer. Studies have shown that the acquisition of epithelial-to-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotypes are highly inter-related, and contributes to drug resistance, tumor recurrence, and metastasis. The molecular mechanism(s) by which FoxM1 contributes to the acquisition of EMT phenotype and induction of CSC self-renewal capacity is poorly understood. Therefore, we established FoxM1 over-expressing pancreatic cancer (AsPC-1) cells, which showed increased cell growth, clonogenicity, and cell migration. Moreover, over-expression of FoxM1 led to the acquisition of EMT phenotype by activation of mesenchymal cell markers, ZEB1, ZEB2, Snail2, E-cadherin, and vimentin, which is consistent with increased sphere-forming (pancreatospheres) capacity and expression of CSC surface markers (CD44 and EpCAM). We also found that over-expression of FoxM1 led to decreased expression of miRNAs (let-7a, let-7b, let-7c, miR-200b, and miR-200c); however, re-expression of miR-200b inhibited the expression of ZEB1, ZEB2, vimentin as well as FoxM1, and induced the expression of E-cadherin, leading to the reversal of EMT phenotype. Finally, we found that genistein, a natural chemo-preventive agent, inhibited cell growth, clonogenicity, cell migration and invasion, EMT phenotype, and formation of pancreatospheres consistent with reduced expression of CD44 and EpCAM. These results suggest, for the first time, that FoxM1 over-expression is responsible for the acquisition of EMT and CSC phenotype, which is in part mediated through the regulation of miR-200b and these processes, could be easily attenuated by genistein.

Figure 1

FoxM1 expression vector increased cell growth (A), the comparative expression of FoxM1 mRNA (B), the relative expression of FoxM1, cyclin D1, VEGF, p65, Hes-1, ZEB1, CD44, and EpCAM at the protein levels (C), and the acquisition of EMT phenotype (D; magnification X 20) in human pancreatic cancer AsPC-1 cells. FoxM1 over-expressing AsPC-1 cells were established by stable gene transfection technique, as described under the Methods section.

Figure 2

FoxM1 over-expression differentially regulated the miRNA expression of let-7a, b, c and miR-200b, c in AsPC-1 cells. The comparative expressions of miRNAs against control RNU6B miRNA were measured by real-time RT-PCR technique.

Figure 3

Re-expression of miR-200b differentially regulated the expression of EMT phenotype marker mRNAs (A) and proteins (B), and reversed EMT phenotype (C; magnification X 20) in FoxM1 over-expressing AsPC-1 cells. Over-expression of miR-200b was established in FoxM1 over-expressing AsPC-1 cells by transient transfection with its precursor. Real-time RT-PCR and Western blotting analysis were performed to measure the relative levels of mRNAs and proteins, respectively.

Figure 4

Genistein treatment inhibited cell survival (A), clonogenicity (B), and the relative levels of expression of proteins (C) in AsPC-1-control and AsPC-1-FoxM1 cells. Different concentrations of genistein were used for both cell lines for 3 days followed by MTT assay, clonogenic assay, and Western blotting analysis, respectively.

Figure 5

Genistein inhibited the capacity of wound healing in AsPC-1-control and AsPC-1-FoxM1 cells. Wound healing assay was conducted to assess the capacity of cell migration and invasion in AsPC-1 cells.

Figure 6

Genistein inhibited the capacity of CSC-self-renewal in primary pancreatospheres of AsPC-1-control and AsPC-1-FoxM1 cells after 1 week of treatment (A) and secondary pancreatospheres of FoxM1 over-expressing AsPC-1 cells after 1 and 3 weeks of treatment (B,C). Secondary pancreatospheres were generated by desegregations of primary pancreatospheres using accutase solution, as described in the Materials and Method section.

Figure 7

Genistein inactivated the expression of CSC surface markers such as CD44 and EpCAM in pancreatospheres of AsPC-1-control and AsPC-1-FoxM1 cells. Immuno-fluorescent staining and confocal microscopy image analysis (Magnification X 250) was conducted using pancreatospheres of AsPC-1-control and AsPC-1-FoxM1 cells after 1 week of 60 µM of genistein treatment.