A miRNA Signature of Chemoresistant Mesenchymal Phenotype Identifies Novel Molecular Targets Associated with Advanced Pancreatic Cancer

<div><p>In this study a microRNA (miRNA) signature was identified in a gemcitabine resistant pancreatic ductal adenocarcinoma (PDAC) cell line model (BxPC3-GZR) and this signature was further examined in advanced PDAC tumor specimens from The Cancer Genome Atlas (TCGA) database. BxPC3-GZR showed a mesenchymal phenotype, expressed high levels of CD44 and showed a highly significant deregulation of 17 miRNAs. Based on relevance to cancer, a seven-miRNA signature (miR-100, miR-125b, miR-155, miR-21, miR-205, miR-27b and miR-455-3p) was selected for further studies. A strong correlation was observed for six of the seven miRNAs in 43 advanced tumor specimens compared to normal pancreas tissue. To assess the functional relevance we initially focused on miRNA-125b, which is over-expressed in both the BxPC3-GZR model and advanced PDAC tumor specimens. Knockdown of miRNA-125b in BxPC3-GZR and Panc-1 cells caused a partial reversal of the mesenchymal phenotype and enhanced response to gemcitabine. Moreover, RNA-seq data from each of 40 advanced PDAC tumor specimens from the TCGA data base indicate a negative correlation between expression of miRNA-125b and five of six potential target genes (<i>BAP1</i>, <i>BBC3</i>, <i>NEU1</i>, <i>BCL2</i>, <i>STARD13</i>). Thus far, two of these target genes, <i>BBC3</i> and <i>NEU1</i>, that are tumor suppressor genes but not yet studied in PDAC, appear to be functional targets of miR-125b since knockdown of miR125b caused their up regulation. These miRNAs and their molecular targets may serve as targets to enhance sensitivity to chemotherapy and reduce metastatic spread.</p></div

Knockdown of micro-RNA-125 reverses the mesenchymal phenotype and increases the drug sensitivity in BxPC3-GZR cells.

<p><b>A.</b> Establish the Lenti-viral based stable cells expressing anti-miR-125b in BxPC3-GZR (Zip-control and miR-125b knock-down). <b>B.</b> TaqMan qPCR assays showing the knockdown of miR-125b in BxPC3-GZR-Zip ctrl cells compared to BxPC3-GZRΔmiR-125b cells. <b>C.</b> Expression of anti-miR-125b (Zip technology, SBI) decreases the expression of EMT and stemness marker monitored by Western bolt assays. Panels <b>D</b> and <b>E</b> show the attenuation of cell migration by knocking down miR-125b expression. Images were taken after crystal violet staining of migrated cells and the data was plotted as a graph (Bar = 50 µm). <b>F.</b> Knockdown of miR-125b increases response to gemcitabine. BxPC3-GZR-ZiP-Ctrl and BxPC3-GZRΔmiR-125b cells were treated with gemcitabine at different concentrations and the MTT assays were performed after 96 hours of treatment. <b>G.</b> Knockdown of miR-125b in Panc-1 cells. Lenti-viral based stable cells were generated to inhibit miR-125b expression (Zip technology,). TaqMan qPCR assays were performed to measure the expression of miR-125b in Zip-control Panc-1 cells and knock down cells (Panc-1 ΔmiR-125b). <b>H.</b> Inhibiting miR-125b decreases CD44 and vimentin expression while up regulating the expression of E-cadherin. <b>I.</b> Knockdown of miR-125b increases the response of Panc-1 to gemcitabine. MTT assays were done after 96 hours of gemcitabine treatment. Statistical significance values *p<0.05 and **p<0.01 were calculated using student's T-tests.</p

Micro-RNA-125b partially regulates CRMP.

<p><b>A.</b> Western blot analyses showing the expression levels of EMT markers in PDAC cells in which miR-125b expression was determined. <b>B.</b> TaqMan qPCR analysis showing the relative expression of miRNAs (miR-125b and miR-30d) in different PDAC cells. MiR-30d is used as a control. <b>C.</b> Induction of miR-125b expression in BxPC3 upon treatment of gemcitabine. Quantitative PCR (TaqMan) were performed after 72 hrs of incubation with the drug in order to monitor the expression level of miR-125b in BxPC3 cells. Data indicates that expression of miR-125b is increased by the treatment of gemcitabine in a dose dependent manner.</p

Characterization of BxPC3-GZR, a cell line model for CRMP.

<p><b>A.</b> Morphological differences between parental BxPC3 and chemoresistance mesenchymal BxPC3-GZR cells. <b>B.</b> Western blot showing that BxPC3-GZR cells possess an EMT phenotype, which is demonstrated by increased expression of vimentin and a decrease of E-cadherin and express the stem cell marker CD44. <b>C.</b> MTT assay comparing the growth of the parental BxPC3 and BxPC3-GZR at 96 hrs after treatment with different concentrations of gemcitabine. <b>D</b>. Western blot comparing the expression of CD44, E-cadherin, Zeb-1 and vimentin after four passages of BxPC3 and BxPC3-GZR. <b>E</b> and <b>F</b>. Immunofluorescence confocal microscopy images show the differential expression of CD44 and vimentin in BxPC3 and BxPC3-GZR cells.</p

Comparison and validation of in vitro data with the patient samples.

<p>TCGA data was analyzed to determine the differential expression of different miRNAs indentified in BxPC3-GZR cells.</p><p>*ND = Not detected (very low copy number). NA = these micro-RNA data are not available in the database.</p><p>Comparison and validation of in vitro data with the patient samples.</p

Gene targets of miR-125b. TCGA data analyses were performed with the clinical pancreatic tumor specimens (n = 40) for miRNA expression with the corresponding target mRNAs.

<p><b>A.</b> Target mRNA expression profile analyses for miR-125b. Determined the expression of known miR-125b targets (<i>BBC3</i> (PUMA), <i>BCL2</i>, <i>STARD13</i>, <i>BAK1</i>, <i>BAP1</i>, <i>ITCH</i> and <i>NEU1</i>). <b>B.</b> A direct co-relation of the target mRNA and miR-125b expression level were measured in the same tumor from pancreatic adenocarcinoma [PDAC] patient's sample. First panel explains the different quadrants which includes up-regulation (+ Ve sign) or down regulation (<b>-</b> Ve sign) of mRNA and miRNA expression levels. Other panels are representing expression level of different mRNA (<i>BBC3, Neu1 and BAK1</i>) compared with the expression of miR-125b in the same tumor.</p

Nuclear translocation of NF-κB sub-unit.

<p><b>A</b>. Western blot analysis for the NF-κB (p65) expression level in different genetically modified HPNE cells. <b>B</b>. Western blot analysis showing constitutive nuclear expression of p65 in cells expressing oncogenic K-Ras and loss of Smad4. <b>C</b>. Effect of EGF on nuclear translocation of NF-κB (p65). HPNE/K-Ras/ShSmad4 cells are treated with EGF (50 ng/ml) and harvested at indicated time points. Western blot analyses were performed with both nuclear and cytoplasmic extracts. A parallel Coomassie blue stained gel was presented as a loading control. <b>D</b>. Nuclear translocation of p65 is partially blocked by treatment of inhibitors described above in Figure 2. <b>E</b>. NF-B mediated regulation of uPA and MMP9. ChIP assay was performed to show the binding of NF-κB to the promoters of uPA and MMP9. The details for ChIP assay and PCR primers are described in material and methods section. </p

The list of miRNAs that are highly significant in terms of expression in the gemcitabine resistant BxPC3-GZR cells compared to control BxPC3 cells.

†<p>These transcripts are statistically significant but very low signal.</p><p>The list of miRNAs that are highly significant in terms of expression in the gemcitabine resistant BxPC3-GZR cells compared to control BxPC3 cells.</p

A working model is illustrating how oncogenic K-Ras and loss of Smad4 cooperate to cause an invasive phenotype.

<p>Oncogenic K-Ras signaling induces EGFR expression and K-Ras induced EGFR expression is normally suppressed by Smad4-dependent TGF-β signaling. Loss of Smad4 therefore leads to optimal up-regulation of K-ras induced EGFR expression. The increased expression and signaling by EGFR activates PI3K which induces nuclear translocation of NF-κB that in turn drives the expression of MMP9 and uPA.</p

miRNA signature of BxPC3-GZR cells.

<p><b>A.</b> Heat map data analysis of the miRNA microarray assays comparing BxPC3 parental cells and drug resistant BxPC3-GZR cells. Only miRNAs were chosen which had significantly over or under expressed as compared to parental cells (high fold changes based on log2 values and very low p-values) were taken for further validation. The statistical values are represented in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106343#pone-0106343-t001" target="_blank"><i>Table 1</i></a>. <b>B.</b> Validation of miRNA microarray data was done for eight differentially expressed miRNAs using TaqMan qPCR assay. In each cell line, the expression level of indicated miRNA was compared between parental BxPC3 cells and BxPC3-GZR cells. RNU43 or U6 was used as an internal miRNA control. <b>C.</b> TCGA data analysis showing that 6 of the 7 miRNAs validated for being deregulated in BxPC3-GZR cells also showed differential expression in tumor specimens from patients with advanced PDAC. Tumor specimens from 43 patients with advanced PDAC were analyzed for miRNA expression compared to normal pancreas tissue.</p