<i>Dicer</i> Is Required for Maintenance of Adult Pancreatic Acinar Cell Identity and Plays a Role in Kras-Driven Pancreatic Neoplasia

<div><p>The role of miRNA processing in the maintenance of adult pancreatic acinar cell identity and during the initiation and progression of pancreatic neoplasia has not been studied in detail. In this work, we deleted <i>Dicer</i> specifically in adult pancreatic acinar cells, with or without simultaneous activation of oncogenic Kras. We found that <i>Dicer</i> is essential for the maintenance of acinar cell identity. Acinar cells lacking <i>Dicer</i> showed increased plasticity, as evidenced by loss of polarity, initiation of epithelial-to-mesenchymal transition (EMT) and acinar-to-ductal metaplasia (ADM). In the context of oncogenic Kras activation, the initiation of ADM and pancreatic intraepithelial neoplasia (PanIN) were both highly sensitive to <i>Dicer</i> gene dosage. Homozygous <i>Dicer</i> deletion accelerated the formation of ADM but not PanIN. In contrast, heterozygous <i>Dicer</i> deletion accelerated PanIN initiation, revealing complex roles for <i>Dicer</i> in the regulation of both normal and neoplastic pancreatic epithelial identity.</p></div

Deletion of <i>Dicer</i> induces loss of polarity in acinar cells following Dicer deletion.

<p>(A) Epithelial cell polarity marker CD49f (white) is expressed basally, while phalloidin labeling (red) is observed apically in control <i>Mist-Cre^ERT2; LSL-YFP; Dicer^wt/wt</i> pancreas following tamoxifen treatment. (B) In the <i>Mist-Cre^ERT2; LSL-YFP; Dicer^fl/fl</i> mice, <i>Dicer</i> deletion leads to translocation of CD49f in the lateral membrane, and loss of apical phalloidin labeling. Cytoplasmic expression of YFP is shown in green as a surrogate marker of Cre-mediated recombination. Nuclei are stained with DAPI (blue).</p

Deletion of <i>Dicer</i> alters histology in exocrine pancreas.

<p>(A) Experimental regimen. Tamoxifen injection was performed on 6–8 weeks old mice. (B) Pancreas 2 months after treatment with corn oil has no YFP expression. (C) Widespread Cre-mediated recombination is shown by YFP expression 2 month following tamoxifen induction. (D, E) Immunohistochemistry for Dicer on <i>Mist-Cre^ERT2; LSL-YFP; Dicer^fl/fl</i> mice 2 month after treatment with corn oil only (D) or those treated with tamoxifen (E). There is almost complete loss of cytoplasmic Dicer signal in the acinar cells in tamoxifen treated tissue. Residual nuclear signal likely represents non-specific antibody labeling. (F–K) H & E staining showing the time-course of histologic change after tamoxifen administration. (F) <i>Dicer^fl/fl</i> Oil control. (G) <i>Dicer^fl/wt</i>. Tissue is morphologically indistinguishable from oil control. (H) <i>Dicer^fl/fl</i> 0.5 month after tamoxifen injection. (I) <i>Dicer^fl/fl</i> 1 month after tamoxifen injection. Tissue changes are most dramatic at this time point. (J) 2 months. (K) 6 months. At this time point, pancreas from Dicer knockout mice is nondistinguishable with wildtype. Scale bars depict 50 microns.</p

<i>Dicer</i> deletion leads to upregulation of ductal markers in the acinar population.

<p>(A, B) Pancreatic ductal marker Dolichos Biflorus Agglutinin (DBA) (purple) is upregulated along with cell proliferation marker Ki67 (red) in the <i>Mist-Cre^ERT2; Dicer^fl/fl</i> mice following tamoxifen injection (B) compared to control <i>Mist-Cre^ERT2; Dicer^wt/wt</i> pancreata (A). (C, D) Sox9 (red), a duct-specific transcription factor, is also upregulated in acinar cells and in ADM lesions in <i>Mist-Cre^ERT2; Dicer^fl/fl</i> pancreata (D), while Sox9 is expressed only in ductal epithelial cells in control <i>Mist-Cre^ERT2; Dicer^wt/wt</i> pancreata (C). Epithelial marker Ecad is in green and nuclei are stained with DAPI (blue).</p

Kras-driven pancreatic lesions are sensitive to Dicer gene dosage.

<p>(A–D) Brown staining indicates immunohistochemical labeling for Dicer (hemotoxylin counterstain, blue). (A) Wildtype pancreas. There is strong cytoplasmic labeling for Dicer in acinar cells. (B) <i>Dicer-wt-Kras</i>. Dicer is down-regulated in acinar cells following oncogenic Kras activation. Notice that PanIN lesions (arrows) have higher Dicer expression. (C) <i>Dicer-floxed-Kras</i>. Residual nuclear signals likely represent non-specific antibody labeling. (D) In the <i>Dicer-floxed-Kras</i> pancreata, areas of ADM retain higher levels Dicer expression. Normal ductal epithelium, stromal cells and endocrine cells (lower right) also show normal expression of Dicer. E–G, H&E labeling demonstrates representative histology (E) <i>Dicer-wt-Kras</i>, (F) <i>Dicer-het-Kras</i> and (G) <i>Dicer-floxed-Kras</i> pancreata. (H) Quantification of surface area occupied by stromal infiltrate, ADM and PanIN. n>5 for each genotype. Error bars indicate standard error. Star denotes comparisons with p-value<0.05.</p

<i>In vitro</i> deletion of <i>Dicer</i> in pancreatic acinar cells results in initiation of EMT.

<p>(A–E) and (K–O) depict multi-channel fluorescent imaging of two representative pancreatic acini independently harvested from <i>Mist1^CreERT2/+</i>; <i>LSL-YFP; Dicer^fl/fl</i> mice and treated with DMSO control. (F–J) and (P–T) depict multi-channel fluorescent imaging of two representative pancreatic acini isolated from <i>Mist1^CreERT2/+</i>; <i>LSL-YFP; Dicer^fl/fl</i> mice and treated with tamoxifen (TMX) to induce Cre activity. (A–J), Immunostaining of isolated acinus with Amylase (red), Ecad (green) and GFP (purple) There is no change in Amylase expression following 5 days incubation with either DMSO control (A–E) or tamoxifen treatment (F–J). But significant down-regulation of Ecad can be observed. (K–T), Immunostaining of isolated acini with Vimentin (red), Ecad (green) and YFP (purple). Down-regulation of Ecad is accompanied by activation of Vimentin in tamoxifen-treated cells (P–T), compared to DMSO control (K–O). Activation of YFP in (H) and (R) document tamoxifen-induced Cre activity.</p

Adult acinar cells undergo epithelial to mesenchymal transition.

<p>(A) Pancreata from oil injected <i>Mist-Cre^ERT2; LSL-YFP; Dicer^fl/fl</i> mice do not express YFP (white) or Vimentin (red). Ecad (green) outlines acinar cells. (B) In tamoxifen treated <i>Mist-CreERT2; LSL-YFP; Dicer^fl/fl</i> mice, acinar cells show co-localization of YFP and Vimentin. Ecad expression is reduced. (C, D) Representative immunofluorescent staining for the mesenchymal marker Ncad (green). (C) In oil treated control pancreas, acini lack any Ncad expression. (D) In tamoxifen treated <i>Dicer^fl/fl</i> pancreas, there is membrane expression of Ncad. Note that there is simultaneous loss of EpCam expression (white), in <i>Dicer</i> deleted acini. Nuclei are stained with DAPI (blue).</p

MUC1.CT directly interacts with transcriptional repressor ZEB1.

<p>A) Co-immunoprecipitation (Co-IP) was utilized to evaluate the interaction of the MUC1.CT and ZEB1 in S2.013.Neo and S2.013.MUC1 cells. Cell lysates were immunoprecipitated with an antibody against the MUC1.CT, and the subsequent western was blotted with an antibody to ZEB1. Non-immunoprecipitated lysates were used as a loading control, and steady state levels of ZEB1 were higher in S2.013.MUC1 cells compared to S2.013.Neo cells. B-C) Proximity ligation assay (PLA) was used to visualize MUC1.CT and ZEB1 interaction in S2.013.Neo and S2.013.MUC1 cells, B, and Panc1.Neo and Panc1.MUC1 cells, C (with representative compressed z-stack image for each). These data show quantitative levels of MUC1.CT and ZEB1 interactions, indicating that MUC1.CT and ZEB1 interact in both cell lines. In the S2.013.Neo and S2.013.MUC1 cell lines, the interaction was significantly higher in the MUC1 expressing cells, confirming the Co-IP in A. However, there was no significant difference in interaction in the Panc1.Neo and Panc1.MUC1 cells. (Student’s t-test: *** p < 0.0005).</p

qRT-PCR confirmation of microRNA levels in cells and pancreatic cancer tissue.

<p>MicroRNA levels were evaluated in triplicate by qRT-PCR to confirm expression changes identified by microarray analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073306#pone-0073306-t001" target="_blank">Table 1</a>). MicroRNA levels were normalized to the U6 RNA control. A) MiRs-200c, -141, -192, -33b, -194, -215, and -376c expression levels were evaluated in S2.013.Neo compared to S2.013.MUC1 cells. B) MiRs-200c, -141, -192, -33b, -194, -215, and -376c expression levels were evaluated in Panc1.Neo and Panc1.MUC1 cells. C) Levels of miR-200c were evaluated in a panel of pancreatic cancer tissues. These data are represented by Ct value, where lower Ct values indicate higher levels of miR-200c, and each data point represents a different patient sample. Uninvolved tissue is non-cancerous tissue isolated from a cancer patient. (Student’s t-test: *** p < 0.0005, ** p < 0.005, * p < 0.05).</p

Chromatin Immunoprecipitation confirmation of MUC1.CT and ZEB1 occupancy of the miR-200c/141 promoter.

<p>Chromatin immunoprecipitation (ChIP) was utilized to confirm MUC1.CT and ZEB1 occupancy at the miR-200c/141 promoter region. A) Schematic representing the miR-200c/141 promoter region where the ChIP primer sets were designed (-480 to -210, and +552 to +675) and the known ZEB1 binding region (Grey bar). B) ChIP results indicate fold change of normalized relative levels of MUC1.CT occupancy at the ZEB1 binding region (based on qPCR) of the miR-200c/141 promoter in S2.013.MUC1 cells compared to S2.013.Neo cells. C) ChIP results indicate fold change of normalized relative levels of ZEB1 occupancy at the ZEB1 binding region (based on qPCR) of the miR-200c/141 promoter in S2.013.MUC1 cells compared to S2.013.Neo cells. D) Sequence of the MUC1 cytoplasmic tail, indicating three phosphorylated tyrosine motifs that were examined here. E-F) ChIP results indicate relative levels (based on qPCR and normalized to IgG control) of phospho-YHPM, -YVPP, and -YEKV MUC1 cytoplasmic tail at the ZEB1 binding region of miR-200c/141 in the S2.013.MUC1 cells, E, and Panc1.MUC1 cells, F. In both cell lines, only the phospho-YEKV form of MUC1.CT shows significant enrichment at the promoter. All ChIP data was normalized to antibody specific IgG control. (Student’s t-test: *** p < 0.0005, ** p < 0.005, * p< 0.05).</p