Identification and Clinical Significance of Pancreatic Cancer Stem Cells and Their Chemotherapeutic Drug Resistance

Abstract

Pancreatic cancer ranks in the 10th–11th position among cancers affecting men in Taiwan, besides being a rather difficult-to-treat disease. The overall 5-year survival rate of pancreatic cancer is only 5–10%, while that of resectable pancreatic cancer is still approximately 15–20%. Cancer stem cells possess intrinsic detoxifying mechanisms that allow them to survive against conventional therapy by developing multidrug resistance. This study was conducted to investigate how to overcome chemoresistance and its mechanisms in pancreatic cancer stem cells (CSCs) using gemcitabine-resistant pancreatic cancer cell lines. Pancreatic CSCs were identified from human pancreatic cancer lines. To determine whether CSCs possess a chemoresistant phenotype, the sensitivity of unselected tumor cells, sorted CSCs, and tumor spheroid cells to fluorouracil (5-FU), gemcitabine (GEM), and cisplatin was analyzed under stem cell conditions or differentiating conditions. Although the mechanisms underlying multidrug resistance in CSCs are poorly understood, ABC transporters such as ABCG2, ABCB1, and ABCC1 are believed to be responsible. Therefore, we measured the mRNA expression levels of ABCG2, ABCB1, and ABCC1 by real-time RT-PCR. Our results showed that no significant differences were found in the effects of different concentrations of gemcitabine on CSCs CD44+/EpCAM+ of various PDAC cell line cultures (BxPC-3, Capan-1, and PANC-1). There was also no difference between CSCs and non-CSCs. Gemcitabine-resistant cells exhibited distinct morphological changes, including a spindle-shaped morphology, the appearance of pseudopodia, and reduced adhesion characteristics of transformed fibroblasts. These cells were found to be more invasive and migratory, and showed increased vimentin expression and decreased E-cadherin expression. Immunofluorescence and immunoblotting experiments demonstrated increased nuclear localization of total β-catenin. These alterations are hallmarks of epithelial-to-mesenchymal transition (EMT). Resistant cells showed activation of the receptor protein tyrosine kinase c-Met and increased expression of the stem cell marker cluster of differentiation (CD) 24, CD44, and epithelial specific antigen (ESA). We concluded that the expression of the ABCG2 transporter protein was significantly higher in CD44+ and EpCAM+ CSCs of PDAC cell lines. Cancer stem-like cells exhibited chemoresistance. Gemcitabine-resistant pancreatic tumor cells were associated with EMT, a more aggressive and invasive phenotype of numerous solid tumors. Increased phosphorylation of c-Met may also be related to chemoresistance, and EMT and could be used as an attractive adjunctive chemotherapeutic target in pancreatic cancer.