The PAX3-FOXO1 Fusion Protein Present in Rhabdomyosarcoma Interferes with Normal FOXO Activity and the TGF-β Pathway

<div><p>PAX3-FOXO1 (PAX3-FKHR) is the fusion protein produced by the genomic translocation that characterizes the alveolar subtype of Rhabdomyosarcoma, a pediatric sarcoma with myogenic phenotype. PAX3-FOXO1 is an aberrant but functional transcription factor. It retains PAX3-DNA-binding activity and functionally overlaps PAX3 function while also disturbing it, in particular its role in myogenic differentiation. We herein show that PAX3-FOXO1 interferes with normal FOXO function. PAX3-FOXO1 affects FOXO-family member trans-activation capability and the FOXO-dependent TGF-β response. PAX3-FOXO1 may contribute to tumor formation by inhibiting the tumor suppressor activities which are characteristic of both FOXO family members and TGF-β pathways. The recognition of this mechanism raises new questions about how FOXO family members function.</p></div

PAX3-FOXO1 inhibits FOXO-transactivation on a FOXO- response element (3XIRS).

<p><b>(A, B and C)</b> 293T cells were co-transfected with a reporter plasmid (pGL2, promega) encoding for firefly luciferase under the control of a FOXO-responsive 3XIRS-containing promoter as well as expression plasmids encoding for the indicated protein (PF: PAX3-FOXO1). 48hours post-transfection, luciferase activity was determined. Where indicated a third plasmid encoding renilla luciferase was included for the control of transfection efficiency. <b>(B)</b> The relative proportion of the FOXO1 and PAX3-FOXO1 plasmids are indicated in brackets. The amount of FOXO1 plasmid was constant with increasing amount of PAX3-FOXO1 plasmid (total amount of transfected DNA was kept constant by using empty expression plasmid). <b>(C)</b> The indicated expression vectors were co-transfected with Empty, PAX3-FOXO1 or PAX3 encoding vectors and transactivation on the 3XIRS containing reporter was evaluated by firefly luciferase measurement normalized to co-transfected renilla luciferase activity. Errors bars represent standard deviation of three experiments.</p

On a bipartite FOXO-SMAD responsive element (4XSFRE), PAX3-FOXO1 inhibits TGF- response and SMAD transactivation.

<p><b>(A)</b> 293T cells were transfected with pGL2-promoter control reporter (pGL2) or a pGL2-promoter reporter containing a bipartite FOXO-SMAD response element (pGL2-4XSFRE) and the indicated expression plasmids (PF: PAX3-FOXO1). In order to avoid transfection efficiency interference, one well of individual transfected cells were trypsinized 24 hours after transfection and splitted in 6 wells. 8 hours later, three wells were left untreated the other three were treated with TGF- (5ng/ml) for further 16hours. Fold induction by TGF- is shown for each transfection. (<b>B)</b> 293T cells were transfected with a pGL2-promoter reporter containing a bipartite FOXO-SMAD response element (pGL2-4XSFRE) and the indicated expression plasmids (PF: PAX3-FOXO1) with or without SMAD3 and SMAD4 expression plasmids. Firefly luciferase activity was determined 48 hours post-transfection and normalized to co-transfected renilla luciferase for correcting for transfection efficiency. <b>(C)</b> 293T cells were transfected with a SMAD binding site-containing reporter, 4XSBE-LUC and the indicated expression plasmids (PF: PAX3-FOXO1). Left panel: TGF- induction was measure. The procedure to avoid transfection efficiency interference was the same as for panel <b>A</b>. Right panel: in addition to the indicated plasmids cells were cotransfected with or without SMAD3 and 4 and fold induction by SMADs was calculated like in panel <b>B</b>. Results of two independent experiments are shown. All experiments were performed at least three times except for panel <b>C</b>. Error bars represent standard deviation of three independent experiments.</p

Ectopic expression of PAX3-FOXO1 interferes with the transcriptional response to TGF-β and affects basal level of expression of FOXO-regulated genes.

<p><b>(A, C, D and F)</b> Indicated cell types were infected with lentiviruses carrying either PAX3-FOXO1 (PF) or GFP coding sequences. Pools of cells were checked for PF or GFP expression and were challenged with TGF-β for 5 hours. Cells were lysed, RNA extracted and levels of indicated mRNAs were measure by real-time PCR and plotted as fold induction by TGF-β. (<b>A)</b> Represents the mean of four experiments; **P<0.01 in two-tailed Student test. (<b>C)</b> Two independent infections and TGF-β inductions, in experiment 1 cells are in MSC medium (Lonza), in experiment 2 in DMEM. (<b>D)</b> Represents the mean of three experiments, **P<0.01 in two-tailed Student test. (<b>F)</b> Represents the means of two experiments. <b>(B</b> and <b>E)</b> the effect that PAX3-FOXO1 exerts on the basal level of expression of the indicated genes is represented as fold induction or repression (if value is negative) compared with the expression level found in GFP-control infected cells. <b>(G)</b> RH30 and RH4 cells were transfected with a control siRNA (siCON) or a siRNA targeting PAX3-FOXO1 (siPF), 32 hours post-transfection cells were challenged with TGF-β for 16 hours and IER3 mRNA levels were measured.</p

siRNA-mediated down regulation of PAX3-FOXO1 in two ARMS cell line restores p15INK4B -TGF-β-response and affects basal level of expression of FOXO-regulated genes.

<p><b>(A and B</b>) siRNA against PAX3-FOXO1 (siPF) or control siRNA (siCONT) were transfected in RH30 and RH4 cell lines. 48 hours after transfection extracts were prepared. (<b>A</b>) Levels of <i>PAX3-FOXO1</i> mRNA were measured by real-time PCR. (<b>B</b>) Levels of PAX3-FOXO1 protein were evaluated by western blot (using an anti-FOXO1 antibody recognizing the C-terminal part of the FOXO1 protein present in PAX3-FOXO1). The position of FOXO1 and PAX3-FOXO1 are indicated. As a control for PAX3-FOXO1, extracts from RD18 cells or RD18 cells infected with a lentivirus carrying PAX3-FOXO1 expression were included. Actin levels were evaluated as loading control. <b>(C)</b> RH30 and RH4 cells were transfected with a control siRNA (siCON) or a siRNA targeting PAX3-FOXO1 (siPF), 32 hours (exp.1) or 48 hours (exp.2) post-transfection cells were challenged with TGF-β for 16 hours (Exp.1) or 7 hours (Exp.2) and <i>p15INK4B</i> mRNA levels were measured by real-time PCR. <b>(D and E)</b> Basal level of expression of indicated genes 40 hours after control (siCON) or PAX3-FOXO1-targetting siRNA (siPF) transfection in RH30 and RH4 cells as measured by real-time PCR. Errors bars represent standard deviation of three independent experiments except for panel C where they represent standard deviation of triplicates in single experiments.</p

ARMS cell lines RH30 and RH4 are poor TGF-β responder.

<p><b>(A)</b> The relative number of viable cells present in the culture after three days of TGFβ treatment (at the indicated concentrations) was evaluated using the colorimetric MTT assay (Millipore). (<b>B and C)</b> Relative levels of <i>p21CIP</i> and <i>p15INK4B</i> mRNAs were evaluated by real-time PCR after treatment with TGF-β. Concentrations of TGF-β and treatment times are indicated. <b>(D and E)</b> Relative mRNA levels of indicated target genes belonging to the FOXO-SMAD synexpression group [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121474#pone.0121474.ref013" target="_blank">13</a>] (<b>D</b>) and genes not belonging to the FOXO-SMAD synexpression group (<b>E</b>). Measurements were performed by real-time PCR after 5 hours of TGF-β treatment at the indicated concentration. <b>(F)</b> Phosphorylation levels of SMAD2 evaluated by western-blot using an anti-phospho-SMAD2 antibody. Cell lines and treatment times (in minutes) are indicated. All experiments were performed at least twice with the exception of panel F. Errors bars represent standard deviation of two experiments in panel A,B,C and three experiments in panel C, D and E.</p

Mesenchymal Stromal Cells Epithelial Transition Induced by Renal Tubular Cells-Derived Extracellular Vesicles

<div><p>Mesenchymal-epithelial interactions play an important role in renal tubular morphogenesis and in maintaining the structure of the kidney. The aim of this study was to investigate whether extracellular vesicles (EVs) produced by human renal proximal tubular epithelial cells (RPTECs) may induce mesenchymal-epithelial transition of bone marrow-derived mesenchymal stromal cells (MSCs). To test this hypothesis, we characterized the phenotype and the RNA content of EVs and we evaluated the <i>in vitro</i> uptake and activity of EVs on MSCs. MicroRNA (miRNA) analysis suggested the possible implication of the miR-200 family carried by EVs in the epithelial commitment of MSCs. Bone marrow-derived MSCs were incubated with EVs, or RPTEC-derived total conditioned medium, or conditioned medium depleted of EVs. As a positive control, MSCs were co-cultured in a transwell system with RPTECs. Epithelial commitment of MSCs was assessed by real time PCR and by immunofluorescence analysis of cellular expression of specific mesenchymal and epithelial markers. After one week of incubation with EVs and total conditioned medium, we observed mesenchymal-epithelial transition in MSCs. Stimulation with conditioned medium depleted of EVs did not induce any change in mesenchymal and epithelial gene expression. Since EVs were found to contain the miR-200 family, we transfected MSCs using synthetic miR-200 mimics. After one week of transfection, mesenchymal-epithelial transition was induced in MSCs. In conclusion, miR-200 carrying EVs released from RPTECs induce the epithelial commitment of MSCs that may contribute to their regenerative potential. Based on experiments of MSC transfection with miR-200 mimics, we suggested that the miR-200 family may be involved in mesenchymal-epithelial transition of MSCs.</p></div

Primers used in qRT-PCR to evaluate microRNAs expression.

<p>Primers used in qRT-PCR to evaluate microRNAs expression.</p

Characterization of EVs protein expression.

<p>(A) Characterization of EVs by cytofluorimetric analysis. Representative FACS analyses of EVs showing the expression of CD24, CD29, CD44, alpha-5 integrin (CD49e), alpha-6 integrin (CD49f), CD73, CD146, EpCAM, and HLA-class I surface molecules and classic exosomal markers CD63, CD81, CD107 (thick lines). Dotted lines indicate the isotypic controls. Three different EV preparations were analyzed with similar results. (B) Representative western blot analysis on RPTEC cells and EVs. Three different experiments were performed with similar results.</p

Primers used in qRT-PCR to evaluate mRNAs expression.

<p>Primers used in qRT-PCR to evaluate mRNAs expression.</p