Activated K-Ras and INK4a/Arf deficiency promote aggressiveness of pancreatic cancer by induction of EMT consistent with cancer stem cell phenotype

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most frequently diagnosed cancers and the fourth leading cause of cancer-related death in the United States, suggesting that there is an urgent need to design novel strategies for achieving better treatment outcome of patients diagnosed with PDAC. Our previous study has shown that activation of Notch and NF-κB play a critical role in the development of PDAC in the compound K-Ras(G12D) and Ink4a/Arf deficient transgenic mice. However, the exact molecular mechanism by which mutated K-Ras and Ink4a/Arf deficiency contribute to progression of PDAC remains largely elusive. In the present study, we used multiple methods, such as real-time RT-PCR, Western blotting assay, and immunohistochemistry to gain further mechanistic insight. We found that the deletion of Ink4a/Arf in K-Ras(G12D) expressing mice led to high expression of PDGF-D signaling pathway in the tumor and tumor-derived cell line (RInk-1 cells). Furthermore, PDGF-D knock-down in RInk-1 cells resulted in the inhibition of pancreatosphere formation and down-regulation of EZH2, CD44, EpCAM, and vimentin. Moreover, we demonstrated that epithelial-mesenchymal transition (EMT) was induced in the compound mice, which is linked with aggressiveness of PDAC. In addition, we demonstrated that tumors from compound transgenic mice have higher expression of cancer stem cell (CSC) markers. These results suggest that the acquisition of EMT phenotype and induction of CSC characteristics could be linked with the aggressiveness of PDAC mediated in part through the activation of PDGF-D, signaling.

Fig. 1

PDGF-D pathway is highly activated in KCI mice. A: The expression of PDGF-D was measured at protein levels in KCI mice by Western blotting analysis. B: PDGF-D and PDGFR-β mRNA expression was detected in KCI mice by real-time RT-PCR technique.

Fig. 2

PDGF-D expression is higher in KCI mice. The PDGF-D expression was detected by IHC in animal tissues. We found that expression of PDGF-D was increased in KCI mice compared with tissues from KC and CI mice.

Fig. 3

PDGF-D siRNA transfection reduces EMT, CSCs markers, and pancreatosphere formation in RInk-1 murine cells. A: PDGF-D siRNA decreased the relative protein levels of Vimentin, EZH2, EpCAM, CD44, and up-regulated E-cadherin. B: It also reduced mRNA levels of EZH2, CD44, and Lin28B, and C: reduced pancreatosphere formation, in RInk-1 cells.

Fig. 4

The existence of stem cell characteristics, and acquisition of EMT in KCI mice. The expression of stem cell markers in the tissues from KC, CI, and KCI mice was detected by real-time RT-PCR and Western blot analysis, respectively. The expression of EMT markers was measured by Western blotting analysis. We found that E-cadherin expression was significantly reduced and the expression of Vimentin and ZEB2 was highly elevated.

Fig. 5

The schematic representation of our proposed mechanism of how PDGF-D promotes the development and progression of PDAC in the KCI mice.