Cysteine cathepsins are rapidly induced and activated in response to c-Myc.

<p>(A) Relative fold mRNA induction of cathepsin B, C, L and S in laser-captured <i>MycER</i>^<i>TAM</i><i>;Bcl-xL</i> pancreatic islets collected at 2, 8, 24 hours and 21 days following c-Myc activation <i>in vivo</i>. Only cathepsin L mRNA is significantly and persistently upregulated following c-Myc activation. *p≤0.05 by one way ANOVA analysis. (B) Cathepsin activity profiles for the pancreatic islets from <i>MycER</i>^<i>TAM</i><i>;Bcl-xL</i> animals using DCG-04 ABP on tissue lysates. The blot shows activity in untreated islets (OFF), which increases following administration of TAM (Myc-ON) for 3 and 7 days. The activity bands corresponding to cysteine cathepsins (CTS) B, L and C are indicated. The data are representative of two independent experiments. Three animals were used for islet purifications at each data point. On the bottom, the graphical representation of cathepsin enzyme activity presented as fold of induction relative to control. The bands intensities were quantified by NIH-FIJI software. The relative expression of each cathepsin tested in the absence of Myc activation is assigned a value 1. (C) Labeling of active proteases <i>in vivo</i>. A cell-permeable fluorescent analog of DCG-04 incorporating the BODIPY fluorophore as a tag, BODIPY 530/550-DCG-04, was injected intravenously into <i>MycER</i>^<i>TAM</i><i>;Bcl-xL</i> and <i>Bcl-xL</i> mice untreated or treated with tamoxifen (TAM) for 7 days. Very little cathepsin activity was observed in islets in non-treated animals (left panel) or Myc-negative control animals injected with TAM for 7 days (right panel). However, <i>MycER</i>^<i>TAM</i><i>;Bcl-xL</i> animals had a profound increase in the cathepsin activity in the Myc-ON hyperplastic islets (middle panel, indicated by arrows). Corresponding images of pancreata counterstained with DNA-binding dye—DAPI are presented. The panels are representatives of at least three animals assayed at each data point, immunohistochemical analyses done in triplicate; seven randomized fields per analysis were considered. Scale bars are 20 μm. The islet area is indicated by the dotted line, I = islet. On the bottom, the quantification of the mean intensity of the BODIFY fluorophore in the islet area quantified by NIH-FIJI software. Statistical analysis was performed using the unpaired Student’s t test.</p

Differentially Expressed miRNAs in Ewing Sarcoma Compared to Mesenchymal Stem Cells: Low miR-31 Expression with Effects on Proliferation and Invasion

<div><p>Ewing sarcoma, the second most common bone tumor in children and young adults, is an aggressive malignancy with a strong potential to metastasize. Ewing sarcoma is characterised by translocations encoding fusion transcription factors with an EWSR1 transactivation domain fused to an ETS family DNA binding domain. microRNAs are post-transcriptional regulators of gene expression and aberrantly expressed microRNAs have been identified as tumor suppressors or oncogenes in most cancer types. To identify potential oncogenic and tumor suppressor microRNAs in Ewing sarcoma, we determined and compared the expression of 377 microRNAs in 40 Ewing sarcoma biopsies, 6 Ewing sarcoma cell lines and mesenchymal stem cells, the putative cellular origin of Ewing sarcoma, from 6 healthy donors. Of the 35 differentially expressed microRNAs identified (fold change >4 and q<0.05), 19 were higher and 16 lower expressed in Ewing sarcoma. In comparisons between Ewing sarcoma samples with EWS-FLI or EWS-ERG translocations, with differing dissemination characteristics and of primary samples and metastases no significantly differential expressed microRNAs were detected using various stringency criteria. For miR-31, the microRNA with lowest expression in comparison to mesenchymal stem cells, functional analyses were performed to determine its potential as a tumor suppressor in Ewing sarcoma. Two of four miR-31 transfected Ewing sarcoma cell lines showed a significantly reduced proliferation (19% and 33% reduction) due to increased apoptosis in one and increased length of G1-phase in the other cell line. All three tested miR-31 transfected Ewing sarcoma cell lines showed significantly reduced invasiveness (56% to 71% reduction). In summary, we identified 35 microRNAs differentially expressed in Ewing sarcoma and demonstrate that miR-31 affects proliferation and invasion of Ewing sarcoma cell lines in ex vivo assays.</p></div

Loss of cathepsin L inhibits Myc-induced tumorigenesis in the <i>MycER</i>^<i>TAM</i><i>;</i>Bcl-xL pancreatic neuroendocrine cancer model.

<p>(A) Histopathological analysis by H&E staining of pancreatic neurodendocrine tumors from the <i>MycER</i>^<i>TAM</i><i>;Bcl-xL</i> and <i>MycER</i>^<i>TAM</i><i>;Bcl-xL;CTSLKO</i> animals untreated (Myc-OFF) or treated with TAM for 14 days (Myc-ON, 14 days). The tumor area is outlined by dotted lines. Images represent pancreata collected from three animals of each genotype. Pancreata collected from animals that retain one functional <i>cathepsin L</i> allele—<i>Myc;Bcl-xL;CTSL</i> HET (<i>CTSL</i>^<i>+/-</i><i>)</i> are presented as a control. Four animals of each genotype were assayed; at least 17 tumors for each genotype were analyzed. Scale bars, 200μm. On the bottom, graph shows quantification of pancreatic islet volume calculated using FIJI software as described in Materials and Methods section. Statistical significance was assessed using the Student T-test). (B) Immunohistochemical analysis of insulin (green) and proliferation marker KI67 (red) in pancreata collected from <i>MycER</i>^<i>TAM</i><i>;Bcl-xL</i> and <i>MycER</i>^<i>TAM</i><i>;Bcl-xL;CTSLKO</i> animals treated as described above. The tumor area is outlined by dotted lines. The asterisks indicate the enlarged areas presented. †—Non-specific staining due to tissue autofluorescence. Three animals of each genotype were assayed; all analyses done in duplicate; six randomized fields per analysis were considered. Scale bars, 100μm.</p

miR-31 expression in mesenchymal stem cells and Ewing sarcomas with different clinicopathological features.

<p>miR-31 expression in 6 MSC samples and 40 ES biopsies was determined using TLDAs. Ct-values were normalized using U6 snRNA. The ES samples were dived into different groups based on tumor size (27 primary tumors, for 6 primary tumors this information was not available) or dissemination characteristics.</p

mRNAs differentially expressed in miR-31 transfected TC-71 cell line compared to negative control.

<p>TC-71 was transfected three times with 100 nM miR-31 or negative control mimics and genome-wide mRNA expression was quantified using Affymetrix GeneChip Human 1.0 ST arrays. Mean values of three independent experiments were used for detection of differentially expressed mRNAs. The 29 genes with a FC >2 and a q-value <0.05 are displayed.</p

Effects of miR-31 on invasiveness of ES cell lines.

<p>Effect of miR-31 on invasion of ES cell lines in ex vivo assays after 72 hours. Invasiveness was analysed in a two chamber cell culture system. After 30 hours or 48 hours cells attached to the membrane in the lower chamber were counted. Shown are average values of three independent experiments. As miR-31 reduced proliferation, cells in additional chambers were treated as the cells used to determine invasion and total cell numbers were counted to calculate a proliferation correction factor with which the numbers of migrated miR-31 transfected cells were multiplied.</p

Loss of cathepsin L does not inhibit onset of Myc-induced tumorigenesis.

<p>(A) Immunohistochemical analysis of endothelial cell proliferation <i>in vivo</i>. Pancreata was isolated from the <i>MycER</i>^<i>TAM</i><i>;Bcl-xL</i> animals from CTS L WT and CTS L-deficient backgrounds untreated or treated with TAM for the duration of three days. Proliferating endothelial cells were identified by co-labeling with the endothelial marker Meca-32 (green) and Ki67 (red). The islet area is outlined by dotted lines. The asterisks indicate the magnified areas of the endothelial compartment of islets presented in the insets. The percentage of Meca-32-positive cells that also stained positive for the proliferation marker Ki67 was then determined as described in Materials and Methods section. At least three animals were assayed of each genotype all analyses done in duplicate; ten randomized fields per analysis were considered. The graph shows the mean and standard error of the mean. ns—no statistical significant difference was detected by Student’s T-test analysis. Scale bars, 25μm. (B) Representative H&E staining of pancreatic sections from <i>MycER</i>^<i>TAM</i>, <i>MycER</i>^<i>TAM</i><i>;Bcl-xL</i> and <i>MycER</i>^<i>TAM</i><i>;Bcl-xL;CTSLKO</i> collected from animals subjected to Myc activation for 3 days. Induction of Myc in the pancreatic islets lacking <i>Bcl-xL</i> expression (<i>Myc</i>) induces profound apoptosis and ablation of the islet beta-cell compartment as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120348#pone.0120348.ref028" target="_blank">28</a>] (right panel). Loss of cathepsin L in <i>MycER</i>^<i>TAM</i><i>;Bcl-xL;CTSLKO</i> (middle panel) has no significant impact on morphology of pancreatic islets following onset of Myc-induced tumorigenesis when compared to <i>MycER</i>^<i>TAM</i><i>;Bcl-xL</i> islets (left panel). At least three animals of each genotype were assayed, eight randomized fields per analysis were considered. Scale bars, 25μm.</p

Myc induces cathepsin L expression in beta-cells of pancreatic Islets.

<p>(A) Immunohistochemical analyses for CTS B, C, L or S expression (all in red) in combination with staining for the pan-leukocyte marker CD45 (green) in pancreatic islet tumors from the <i>MycER</i>^<i>TAM</i><i>;Bcl-xL</i> animals. Pancreata were harvested from the <i>MycER</i>^<i>TAM</i><i>;Bcl-xL</i> mice treated for 7 d with TAM (Myc-On, 7 days) or control vehicle in place of TAM (Myc-OFF). The islet area is indicated by dotted line. The asterisks indicate the area of tumor represented in the insets. The panels are representatives of at least three animals assayed at each data point, all immunohistochemical analyses were done in duplicate; eight randomized fields per analysis were examined. Scale bars, 100μm. (B) Immunohistochemical analysis for cathepsin L expression in beta-cells of pancreatic islets from <i>MycER</i>^<i>TAM</i><i>;Bcl-xL</i> animals identified by insulin expression. Pancreata were collected from the animals described above. Scale bars represent 25μm. The panels are representatives of three animals assayed at each data point, all immunohistochemical analyses were done in duplicate; ten randomized fields per analysis were examined.</p

Loss of cathepsin L in Myc-induced pancreatic neuroendocrine tumors is associated with the elevated expression of markers of cell autophagy and apoptosis <i>in vivo</i>.

<p>(A) Immunohistochemical analysis of LC 5 expression in the pancreata collected from the <i>MycER</i>^<i>TAM</i><i>;Bcl-x</i>_<i>L</i> and <i>MycER</i>^<i>TAM</i><i>;Bcl-x</i>_<i>L</i><i>;CTSLKO</i> animals untreated (Myc-OFF) or treated with TAM for the duration of 14 days (Myc-ON (14 days)). At least four animals were assayed for each genotype; immunohistochemical analyses done in duplicate; ten randomized fields per analysis were considered. The asterisks indicate areas of tumor tissue enlarged in the inset in the upper right corner. Note punctate LC3 staining in the <i>MycER</i>^<i>TAM</i><i>;Bcl-x</i>_<i>L</i><i>;CTSLKO</i> animals. The graph shows quantification of LC3 puncta per cells in described tumors (n = 4 (Myc; BclXL mice) and n = 3 (Myc, Bcl Xl, CTSL mcie) at least eight independent fields for animal were considered). Statistical analysis was done by unpaired Student’s t test. Scale bars, 50 μm. (B) Immunohistochemical analysis of intracellular localization of LC-3 and the lysosomal marker LAMP1 in pancreata collected from the <i>MycER</i>^<i>TAM</i><i>;Bcl-x</i>_<i>L</i> and <i>MycER</i>^<i>TAM</i><i>;Bcl-x</i>_<i>L</i><i>;CTSLKO</i> animals treated with TAM for the duration of 14 days determined by confocal microscopy (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120348#sec009" target="_blank">Materials and Methods</a>). White arrows marked the arrears of co-localization of LC3 and LAMP1 staining. At least three animals were assayed for each genotype; immunohistochemical analyses done in duplicate; nine randomized fields per analysis were considered. Graph shows LC3+ and LAMP1+ co-localization presented as the percentage of total LC3+ dots. *** p<0.0001 by the two-tailed Student’s t test. (C) Representative images from an IHC analysis of apoptosis assayed by activated caspase-3 (CC-3) staining in pancreata collected from the the <i>MycER</i>^<i>TAM</i><i>;Bcl-x</i>_<i>L</i> and <i>MycER</i>^<i>TAM</i><i>;Bcl-x</i>_<i>L</i><i>;CTSLKO</i> animals treated with control vehicle (Myc-OFF) or TAM for the duration of 1, 3 or 14 consecutive days. Islet area is outlined by dotted line. At least three animals were assayed for each genotype and time point. Graph shows quantification of CC3+ cells in islet area. Scale bars = 50 μm.</p

Unsupervised hierarchical clustering of miRNA expression profiles of ES and MSCs.

<p>miRNA expression profiles of 52 samples, 6-values were normalized using U6 snRNA to generate relative expression levels. Unsupervised cluster analysis was based on Pearson’s correlation (unweighted average) and performed without any stringent filtering criteria. The samples are separated in two branches, MSC and ES samples and the ES samples are further separated into cell lines and biopsies.</p