Normal kidney development in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A-B: H&E stained kidney sections from day E17.5 showing normal development both in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice and controls (X200).</p

Disorganization of extracellular matrix and blood vessels in the lungs of <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A-F: Representative images of immunohistochemistry for pan-laminin (X400) (<b>A,B</b>), and immunofluorescence for entactin (X630) (<b>C,D</b>), showed a different pattern in E17.5 lung from <i>UPK II-Cre;LSL-K-ras^G12D</i> mice, with a less organized network and stronger expression in the stroma. Immunofluorescence for collagen IV (X630) (<b>E,F</b>) showed a similar, but less prominent, pattern. <b>G-J</b>: CD34 staining (<b>G,H</b>) and CD31 immunofluorescence (<b>I,J</b>) of lung vessels at E17.5 (X400) also exhibited a disorganized distribution in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice, with more mesenchymal vessels and a disruption of the normal subepithelial double capillary network (black arrows in <b>G</b>).</p

Lung phenotype of <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A–B: Representative H&E stained sections showing normality of lung (X200) morphology in <i>UPK II-Cre;LSL-K-ras^G12D</i> when compared with controls at E14.5 (pseudoglandular stage). <b>C–H</b>: Formation of air spaces was impaired in lungs (X100) of <i>UPK II-Cre;LSL-K-ras^G12D</i> mice at E17.5 (<b>C,D</b>) and E19.5 (<b>E,F</b>), reflecting a defective septation process. P1 lung morphology was also different between controls and transgenic mice, which exhibited an enlargement and simplification of sacculi (<b>G,H</b>). <b>I–J</b>: Morphometric analysis of lung sections showed a decreased total air space (<b>I</b>) and mean alveolar (saccular) area (<b>J</b>) in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice at E17.5 (n = 5); the impairment of air space development led to an increased total air space area and mean alvelolar area in the early postnatal period (P1; n = 12) in transgenic mice when compared to controls; bars, SEM. <b>K–L</b>: Masson tri-chrome staining of day P1 <i>UPK II-Cre;LSL-K-ras^G12D</i> lungs (X100), showing absence of fibrosis both in cases and controls. <b>M</b>: The direct expression of K<i>-ras^G12D</i> was ruled out with specific PCR showing the absence of recombination between <i>K-ras</i> and the Lox sequence in lung and placenta.</p

Characterization of the <i>UPK II-Cre;Rosa-Stop-YFP</i> reporter mice.

<p>A–J: <i>UPK II-Cre;Rosa-Stop-YFP^+/+</i> reporter mice reveal fluorescence only in the bladder urothelium (x200) (<b>A,B</b>) and ureter urothelium (x200) (day 1, P1) (white arrow) (<b>C,D</b>), but not in lung (x200) (P1) (<b>E,F</b>), placenta (x200) (E19.5) (<b>G,H</b>) or yolk sac (x200) (E19.5) (<b>I,J</b>) among other negative tissues. <b>K</b>: PCR for recombinant UPK II-YFP was only positive in <i>UPK II-Cre;Rosa-Stop-YFP^+/+</i> bladder (E19.5).</p

Fragmentation of ECM components in lungs of <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A: Total protein lysates from whole lung were analyzed by Western blot for for laminin β-1 from P1 <i>UPK II-Cre;LSL-K-ras^G12D</i> mice and control (Cre-) mice, showing an additional low molecular weight (mw) band (35 kDa) which suggest fragmentation. <b>B</b>: Representative images of immunofluorescence for laminin β-1 (X200) showed a disorganized membrane pattern in E17.5 lung from <i>UPK II-Cre;LSL-K-ras^G12D</i> mice. <b>C</b>: WB for lung E-cadherin in P1 lung, with an increase in low mw bands. (53 and 32 KDa) also in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.</p

Urothelial hyperplasia in the <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A: Urothelial-restricted expression of K-ras^G12D. <b>B–E</b>: H&E analysis of bladders (X200) reveals a hyperplastic urothelium at E17.5 (<b>B,C</b>) and P1 (<b>D,E</b>) (black arrows). <b>F</b>: Differences in urothelial cellularity (cells/0.15 mm²) between <i>UPK II-Cre;LSL-K-ras^G12D</i> mice and controls were significant both at E17.5 (n = 2/group; *, <i>P</i> = 0.05) and P1 (n>6/group; **, <i>P</i><0.0001); bars, SEM. <b>G–H</b>: BRDU staining of bladder (X200) showing a higher proliferation in E17.5 <i>UPK II-Cre;LSL-K-ras^G12D</i> mice (urothelium limit is marked with a blue line). The mean number of BrdU positive nuclei/200 µm of urothelium was significantly higher than in controls (n = 10; 6±2 positive nuclei/200 µm vs 1.33±0.81; <i>P</i> = 0.01).</p

Angiogenic role of miR-20a in breast cancer

<div><p>Background</p><p>Angiogenesis is a key process for tumor progression and a target for treatment. However, the regulation of breast cancer angiogenesis and its relevance for clinical resistance to antiangiogenic drugs is still incompletely understood. Recent developments on the contribution of microRNA to tumor angiogenesis and on the oncogenic effects of miR-17-92, a miRNA cluster, point to their potential role on breast cancer angiogenesis. The aim of this work was to establish the contribution of miR-20a, a member of miR-17-92 cluster, to tumor angiogenesis in patients with invasive breast carcinoma.</p><p>Methods</p><p>Tube-formation in vitro assays with conditioned medium from MCF7 and MDA-MB-231 breast cancer cell lines were performed after transfection with miR-20a and anti-miR20a. For clinical validation of the experimental findings, we performed a retrospective analysis of a series of consecutive breast cancer patients (n = 108) treated with neoadjuvant chemotherapy and with a full characterization of their vessel pattern and expression of angiogenic markers in pre-treatment biopsies. Expression of members of the cluster miR-17-92 and of angiogenic markers was determined by RT-qPCR after RNA purification from FFPE samples.</p><p>Results</p><p><i>In vitro</i> angiogenesis assays with endothelial cells and conditioned media from breast cancer cell lines showed that transfection with anti-miR20a in MDA-MB-231 significantly decreased mean mesh size and total mesh area, while transfection with miR-20a in MCF7 cells increased mean mesh size. MiR-20a angiogenic effects were abrogated by treatment with aflibercept, a VEGF trap. These results were supported by clinical data showing that mir-20a expression was higher in tumors with no estrogen receptor or with more extensive nodal involvement (cN2-3). A higher miR-20a expression was associated with higher mean vessel size (<i>p</i> = 0.015) and with an angiogenic pattern consisting in larger vessels, higher VEGFA expression and presence of glomeruloid microvascular proliferations (<i>p</i><0.001). This association was independent of tumor subtype and VEGFA expression.</p><p>Conclusions</p><p>Transfection of breast cancer cells with miR-20a induces vascular changes in endothelial tube-formation assays. Expression of miR-20a in breast invasive carcinomas is associated with a distinctive angiogenic pattern consisting in large vessels, anomalous glomeruloid microvascular proliferations and high VEGFA expression. Our results suggest a role for miR-20a in the regulation of breast cancer angiogenesis, and raise the possibility of its use as an angiogenic biomarker.</p></div

Angiogenic <i>in vitro</i> effects of miR-20a in breast cancer cell lines (MCF7 and MDA-MB-231).

<p>Angiogenic <i>in vitro</i> effects of miR-20a in breast cancer cell lines (MCF7 and MDA-MB-231).</p

Effect of VEGFA exposure on miR-20a expression by MCF7 cells.

<p>No significant change of MCF7 miR-20a expression (relative to snU6 expression) after exposure to low (0.5 ng/mL) or high (10 ng/mL) recombinant human VEGFA concentrations (24 and 48 hours).</p

Association of miR-20a expression with vascular characteristics of breast cancer.

<p>(A) Association between high (over the median value) MVS and miR-20a expression in breast cancer (U Mann-Whitney; p = 0.013). (B) Association of high miR-20a (<i>p</i><0.001) and VEGFA (<i>p</i> = 0.002) expression with a high-risk angiogenic profile (GMP+/MVS high).</p