MicroRNA Profiling Implies New Markers of Gemcitabine Chemoresistance in Mutant p53 Pancreatic Ductal Adenocarcinoma.

No reliable predictors of susceptibility to gemcitabine chemotherapy exist in pancreatic ductal adenocarcinoma (PDAC). MicroRNAs (miR) are epigenetic gene regulators with tumorsuppressive or oncogenic roles in various carcinomas. This study assesses chemoresistant PDAC for its specific miR expression pattern.Gemcitabine-resistant variants of two mutant p53 human PDAC cell lines were established. Survival rates were analyzed by cytotoxicity and apoptosis assays. Expression of 1733 human miRs was investigated by microarray and validated by qRT-PCR. After in-silico analysis of specific target genes and proteins of dysregulated miRs, expression of MRP-1, Bcl-2, mutant p53, and CDK1 was quantified by Western blot.Both established PDAC clones showed a significant resistance to gemcitabine (p<0.02) with low apoptosis rate (p<0.001) vs. parental cells. MiR-screening revealed significantly upregulated (miR-21, miR-99a, miR-100, miR-125b, miR-138, miR-210) and downregulated miRs (miR-31*, miR-330, miR-378) in chemoresistant PDAC (p<0.05). Bioinformatic analysis suggested involvement of these miRs in pathways controlling cell death and cycle. MRP-1 (p<0.02) and Bcl-2 (p<0.003) were significantly overexpressed in both resistant cell clones and mutant p53 (p = 0.023) in one clone.Consistent miR expression profiles, in part regulated by mutant TP53 gene, were identified in gemcitabine-resistant PDAC with significant MRP-1 and Bcl-2 overexpression. These results provide a basis for further elucidation of chemoresistance mechanisms and therapeutic approaches to overcome chemoresistance in PDAC

Establishment of chemoresistant PDAC cell clones.

<p>Generation of chemoresistant PDAC cell clones by repeated pulsatile treatment over 3 days with constant sublethal concentrations of 0.4μM (A) or 0.06μM (D) gemcitabine followed by recovery-periods and quantification of cell viability by MTT assay as well as apoptosis assay in parental vs. chemoresistant PANC-1 (A, B, C) and MIA-PaCa-2 (D, E, F) cell clones.</p

Protein target expression.

<p>Protein expression of MRP-1, mutant p53 (mt p53 R273H and mt p53 R248W), CDK1, Bcl-2, and actin (loading control) in parental (PANC-1, MIA-PaCa-2) and gemcitabine resistant PDAC cell clones (PANC-1-GR, MIA-PaCa-2-GR) by Western blot.</p

MicroRNA profiling.

<p>Procedure and results of miR expression profiling by GeneChip microarray and qRT-PCR validation in parental and gemcitabine resistant PANC-1 and MIA-PaCa-2 cell clones.</p

Protein densitometry.

<p>Adjusted relative density of MRP-1, mutant p53 (mt p53 R273H and mt p53 R248W), CDK1, Bcl-2, and their loading controls measured by ImageJ densitometry software. Asterisks indicates to a significant difference of p<0.05, respectively.</p

Intrinsic drug sensitivity of parental PDAC cell lines.

<p>Relative cell viability of five human PDAC cell lines following 72 h exposure to ascending concentrations of gemcitabine by MTT cytotoxicity assay.</p

Morphologic changes in parental and chemoresistant PANC-1 and MIA-PaCa-2.

<p>Representative attached epithelial cells with spindle-shaped cells in untreated MIA-PaCa-2 (A) and PANC-1 cells (B) compared to more plump rounded morphology and enhanced formation of pseudopodia in their chemoresistant cell clones (C, D).</p

In-silico target analysis.

<p>Overview of selected and highly predicted gene targets of dysregulated miRs in chemoresistant PDAC cell clones. (N.A., not available)</p

Interaction of mutant TP53 and microRNA signaling pathways in chemoresistant pancreatic cancer.

<p>Target analysis was performed by Ingenuity Pathway Analysis and DIANA-mirPath.</p

MicroRNA microarray validation.

<p>MiR microarray validation with qRT-PCR for significantly dysregulated miRs in PANC-1-GR (A) and MIA-PaCa-2-GR cell clones (B). Fold change from miR microarray is demostrated by log2 values (left y-axis, dark grey bars). Fold change from qRT-PCR was determined using the 2^-ΔΔCt method (right y-axis, light grey bars). Error bars represent the standard deviation of mean.</p