Epithelial to mesenchymal transition contributes to drug resistance in pancreatic cancer

Abstract

A better understanding of drug resistance mechanisms is required to improve outcomes in patients with pancreatic cancer. Here, we characterized patterns of sensitivity and resistance to three conventional chemotherapeutic agents with divergent mechanisms of action [gemcitabine, 5-fluorouracil (5-FU), and cisplatin] in pancreatic cancer cells. Four (L3.6pl, BxPC-3, CFPAC-1, and SU86.86) were sensitive and five (PANC-1, Hs766T, AsPC-1, MIAPaCa-2, and MPanc96) were resistant to all three agents based on GI(50) (50% growth inhibition). Gene expression profiling and unsupervised hierarchical clustering revealed that the sensitive and resistant cells formed two distinct groups and differed in expression of specific genes, including several features of "epithelial to mesenchymal transition" (EMT). Interestingly, an inverse correlation between E-cadherin and its transcriptional suppressor, Zeb-1, was observed in the gene expression data and was confirmed by real-time PCR. Independent validation experiment using five new pancreatic cancer cell lines confirmed that an inverse correlation between E-cadherin and Zeb-1 correlated closely with resistance to gemcitabine, 5-FU, and cisplatin. Silencing Zeb-1 in the mesenchymal lines not only increased the expression of E-cadherin but also other epithelial markers, such as EVA1 and MAL2, and restored drug sensitivity. Importantly, immunohistochemical analysis of E-cadherin and Zeb-1 in primary tumors confirmed that expression of the two proteins was mutually exclusive (P = 0.012). Therefore, our results suggest that Zeb-1 and other regulators of EMT may maintain drug resistance in human pancreatic cancer cells, and therapeutic strategies to inhibit Zeb-1 and reverse EMT should be evaluated.

Figure 1

Effect of chemotherapeutic drugs on pancreatic cancer cells. A: Proapoptotic effects of gemcitabine on pancreatic cancer cells. 1.0×10⁶ of pancreatic cancer cells were seeded in culture plates, and incubated with and without 10 μM gemcitabine. After 72 hours, apoptotic cells were measured by FACS analysis. Data shown are mean ± SEM from three experiments. B&C: Effects of various chemotherapeutic drugs on pancreatic cancer cell viability. Cells were treated with increasing concentrations of gemcitabine, 5-FU, and cisplatin (1–100,000 nM). After 72 hours, viable cells were quantified using the MTS reagent. Data shown are mean ± SEM from three experiments. D: log₁₀ (GI₅₀) values and cluster analysis of using log₁₀ (GI₅₀) values were plotted. If log₁₀ (GI₅₀) of the cell lines were below the mean of log₁₀ (GI₅₀) across the panel of cells, the cell lines were defined as sensitive, whereas resistant cell lines had log₁₀ (GI₅₀) values above the mean.

Figure 2

Differentially expressed genes between sensitive and resistant cells. A: Unsupervised hierarchical clustering using whole genome expression patterns in the array (average linkage clustering). Cell lines are separated as two distinct subgroups that correlated with drug sensitivity (Blue box: sensitive cells, Red box: resistant cells). B: EMT related genes with significant differences in expression between sensitive and resistant cells (p<0.001). Highly expressed genes are in red and lower expressed genes are in green. C: The differential expression of MAL2, S100A4 and EVA1 were confirmed by real-time PCR. Mean ± SEM from triplicate samples.

Figure 3

Expression of EMT markers in pancreatic cancer cells. A: Expression patterns of E-cadherin, Zeb-1 and Vimentin in the array data were generated via heat map and confirmed by quantitative real-time PCR. An inverse correlation between E-cadherin and Zeb-1 was observed across the cell lines. Mean ± SEM from triplicate samples. B- D: Immunofluorescence localization of E-cadherin, Zeb-1 and Vimentin confirms the association of epithelial and mesenchymal phenotype in drug sensitivity and resistance.

Figure 4

Effects of silencing Zeb-1 on EMT-related gene expression and drug sensitivity. A: Cells were transiently transfected with Zeb-1 specific siRNA or non-specific control for 48 h, and the expression of Zeb-1 and E-cadherin protein were measured in Panc-1 cells. B. The mRNA expression of Zeb-1, E-cadherin, EVA1 and MAL2 were measured after Zeb-1 silencing. Zeb-1-specific siRNA significantly reduced the expression of Zeb-1 and this was associated with a significant induction of E-cadherin, EVA1 and MAL2 mRNA levels. Mean ± SEM from triplicate samples. C: Effects of Zeb-1 silencing on drug sensitivity in the resistant cells. Zeb-1 specific siRNA or non-specific control transfected Panc-1, MIAPaCa-2 and Hs766T cells were incubated with 10 μM gemcitabine, 5FU and cisplatin for up to 72 hrs, then apoptotic cells were measured by PI-FACS analysis. Data converted into percentage of apoptosis over the control.

Figure 5

A: The expression of E-cadherin and Zeb-1 in primary patient tumors. a and b: E-cadherin positive cancer cells are negative for Zeb-1. c and d: Zeb-1 positive cells are negative for E-cadherin. B: The inverse correlation between E-cadherin and Zeb-1 expression in independent validation experiment. C: Apoptotic effects of gemcitabine, cisplatin and 5-FU in a validation set of 5 new cell lines measured by PI-FACS analysis.