Egr2::cre mediated conditional ablation of dicer disrupts histogenesis of mammalian central auditory nuclei.

Histogenesis of the auditory system requires extensive molecular orchestration. Recently, Dicer1, an essential gene for generation of microRNAs, and miR-96 were shown to be important for development of the peripheral auditory system. Here, we investigated their role for the formation of the auditory brainstem. Egr2::Cre-mediated early embryonic ablation of Dicer1 caused severe disruption of auditory brainstem structures. In adult animals, the volume of the cochlear nucleus complex (CNC) was reduced by 73.5%. This decrease is in part attributed to the lack of the microneuronal shell. In contrast, fusiform cells, which similar to the granular cells of the microneural shell are derived from Egr2 positive cells, were still present. The volume reduction of the CNC was already present at birth (67.2% decrease). The superior olivary complex was also drastically affected in these mice. Nissl staining as well as Vglut1 and Calbindin 1 immunolabeling revealed that principal SOC nuclei such as the medial nucleus of the trapezoid body and the lateral superior olive were absent. Only choline acetyltransferase positive neurons of the olivocochlear bundle were observed as a densely packed cell group in the ventrolateral area of the SOC. Mid-embryonic ablation of Dicer1 in the ventral cochlear nucleus by Atoh7::Cre-mediated recombination resulted in normal formation of the cochlear nucleus complex, indicating an early embryonic requirement of Dicer1. Quantitative RT-PCR analysis of miR-96 demonstrated low expression in the embryonic brainstem and up-regulation thereafter, suggesting that other microRNAs are required for proper histogenesis of the auditory brainstem. Together our data identify a critical role of Dicer activity during embryonic development of the auditory brainstem

The proapoptotic gene interferon regulatory factor-1 mediates the antiproliferative outcome of paired box 2 gene and tamoxifen

Abstract Tamoxifen is the most prescribed selective estrogen receptor (ER) modulator in patients with ER-positive breast cancers. Tamoxifen requires the transcription factor paired box 2 protein (PAX2) to repress the transcription of ERBB2/HER2. Now, we identified that PAX2 inhibits cell growth of ER+/HER2− tumor cells in a dose-dependent manner. Moreover, we have identified that cell growth inhibition can be achieved by expressing moderate levels of PAX2 in combination with tamoxifen treatment. Global run-on sequencing of cells overexpressing PAX2, when coupled with PAX2 ChIP-seq, identified common targets regulated by both PAX2 and tamoxifen. The data revealed that PAX2 can inhibit estrogen-induced gene transcription and this effect is enhanced by tamoxifen, suggesting that they converge on repression of the same targets. Moreover, PAX2 and tamoxifen have an additive effect and both induce coding genes and enhancer RNAs (eRNAs). PAX2–tamoxifen upregulated genes are also enriched with PAX2 eRNAs. The enrichment of eRNAs is associated with the highest expression of genes that positivity regulate apoptotic processes. In luminal tumors, the expression of a subset of these proapoptotic genes predicts good outcome and their expression are significantly reduced in tumors of patients with relapse to tamoxifen treatment. Mechanistically, PAX2 and tamoxifen coexert an antitumoral effect by maintaining high levels of transcription of tumor suppressors that promote cell death. The apoptotic effect is mediated in large part by the gene interferon regulatory factor 1. Altogether, we conclude that PAX2 contributes to better clinical outcome in tamoxifen treated ER-positive breast cancer patients by repressing estrogen signaling and inducing cell death related pathways

Loss of progesterone receptor is associated with distinct tyrosine kinase profiles in breast cancer

Purpose The aim of this study was to assess protein tyrosine kinase profiles in primary breast cancer samples in correlation with the distinct hormone and growth receptor profiles ER, PR, and HER2. Experimental design Pamchip® microarrays were used to measure the phosphorylation of 144 tyrosine kinase substrates in 29 ER+ breast cancer samples and cell lines MCF7, BT474 and ZR75-1. mRNA expression data from the METABRIC cohort and publicly available PR chip-sequencing data were used for validation purposes, together with RT-PCR. Results In ER+ breast tumors and cell lines, we observed that the loss of PR expression correlated to higher kinase activity in samples and cell lines that were HER2−. A number of kinases, representing mostly proteins within the PI3K/AKT pathway, were identified as responsible for the differential phosphorylation between PR− and PR+ in ER+/HER2− tumors. We used the METABRIC cohort to analyze mRNA expression from 977 ER+/HER2− breast cancers. Twenty four kinase-encoding genes were identified as differentially expressed between PR+ and PR−, dividing ER+/HER2− samples in two distinct clusters with significant differences in survival (p < 0.05). Four kinase genes, LCK, FRK, FGFR4, and MST1R, were identified as potential direct targets of PR. Conclusions Our results suggest that the PR status has a profound effect on tyrosine kinases, especially for FGFR4 and LCK genes, in ER+/HER2− breast cancer patients. The influence of these genes on the PI3K/AKT signaling pathway may potentially lead to novel drug targets for ER+/PR− breast cancer patients

Loss of progesterone receptor is associated with distinct tyrosine kinase profiles in breast cancer

Purpose The aim of this study was to assess protein tyrosine kinase profiles in primary breast cancer samples in correlation with the distinct hormone and growth receptor profiles ER, PR, and HER2. Experimental design Pamchip® microarrays were used to measure the phosphorylation of 144 tyrosine kinase substrates in 29 ER+ breast cancer samples and cell lines MCF7, BT474 and ZR75-1. mRNA expression data from the METABRIC cohort and publicly available PR chip-sequencing data were used for validation purposes, together with RT-PCR. Results In ER+ breast tumors and cell lines, we observed that the loss of PR expression correlated to higher kinase activity in samples and cell lines that were HER2−. A number of kinases, representing mostly proteins within the PI3K/AKT pathway, were identified as responsible for the differential phosphorylation between PR− and PR+ in ER+/HER2− tumors. We used the METABRIC cohort to analyze mRNA expression from 977 ER+/HER2− breast cancers. Twenty four kinase-encoding genes were identified as differentially expressed between PR+ and PR−, dividing ER+/HER2− samples in two distinct clusters with significant differences in survival (p < 0.05). Four kinase genes, LCK, FRK, FGFR4, and MST1R, were identified as potential direct targets of PR. Conclusions Our results suggest that the PR status has a profound effect on tyrosine kinases, especially for FGFR4 and LCK genes, in ER+/HER2− breast cancer patients. The influence of these genes on the PI3K/AKT signaling pathway may potentially lead to novel drug targets for ER+/PR− breast cancer patients

Malformed CNC in P0 <i>Dicer1^Egr2</i> mice.

<p><b><i>A,B,</i></b> Nissl stained sections of the cochlear nucleus complex of wild type (<b><i>A</i></b>) or <i>Dicer1^Egr2</i> mice aged P0 (<b><i>B</i></b>) at anterior (<b><i>A1,B1</i></b>), middle (<b><i>A2,B2</i></b>) or posterior (<b><i>A3,B3</i></b>) levels. All three subnuclei, i.e. the AVCN, PVCN, and DCN display a reduced size. AVCN, anteroventral cochlear nucleus; DCN, dorsal cochlear nucleus; PVCN, posterior ventral cochlear nucleus. Dorsal is up, lateral is to the right.</p

Severe disruption of the SOC in <i>Dicer1^Egr2</i> mice.

<p><b><i>A–F</i></b> Vglut1 immunoreactivity (<b><i>A,B</i></b>), Calb1 immunoreactivity (<b><i>C,D</i></b>), or Nissl stained sections (<b><i>E,F</i></b>) in the superior olivary complex of adult (P22– P30) wild type (<b><i>A,C,E</i></b>) or <i>Dicer1^Egr2</i> mice (<b><i>B,D,F</i></b>). Only weak Vglut1 staining is observed in the SOC of <i>Dicer1^Egr2</i> mice and principal nuclei such as the MNTB or the U-shaped LSO cell group are absent in these animals. Calb1 labeling is observed in somata of MNTB neurons and in the neuropil of the LSO, MSO, and SPN in wild type mice. No labeling is observed in <i>Dicer1^Egr2</i> mice. In Nissl stained sections, the cell groups of the MNTB and LSO are recognizable in control mice, but not in <i>Dicer1^Egr2</i> mice. The cell group at the ventral part in <i>Dicer1^Egr2</i> mice corresponds to the olivocochlear neurons. <b><i>G–J</i></b> Vglut1 immunoreactivity (<b><i>G,H</i></b>), or Nissl stained sections (<b><i>I,J</i></b>) in the superior olivary complex of P0 wild type (<b><i>G,I</i></b>) or <i>Dicer1^Egr2</i> mice (<b><i>H,J</i></b>). Vglut1 staining is restricted to the olivocochlear neurons in the SOC of <i>Dicer1^Egr2</i> mice, and principal nuclei such as the MNTB or the LSO cell group are absent in these animals. In Nissl stained sections, the cell groups of the MNTB and LSO are recognizable in wild type mice, but not in <i>Dicer1^Egr2</i> mice. The cell group at the ventral part in <i>Dicer1^Egr2</i> mice corresponds to the olivocochlear neurons. LSO, lateral superior olive; MNTB, medial nucleus of the trapezoid body; MSO, medial superior olive; OCN, olivocochlear neurons; SPN, superior paraolivary nucleus. Dorsal is up, lateral is to the right.</p

Reduced size of the nuclei of the lateral lemniscus is attributable to secondary effects of <i>Dicer1</i> loss.

<p><b><i>A–D</i></b>, <i>Egr2::Cre</i> mice were crossed with <i>ROSA26R</i> mice, resulting in expression of β-galactosidase after Cre-mediated recombination. Only few X-gal positive cells were detected in the nuclei of the lateral lemniscus in the overview (<b><i>A,B</i></b>) or at high magnification (<b><i>C,D</i></b>). <b><i>E,F,</i></b> Vglut1 labeling of the nuclei of the lateral lemniscus in adult (P29, 2 animals per genotype) wild type (<b><i>E</i></b>) or <i>Dicer1^Egr2</i> mice (<b><i>F</i></b>) revealed reduced size of the nuclei in <i>Dicer1^Egr2</i> mice. <b><i>G,H</i></b>, Diminished size of the nuclei in <i>Dicer1^Egr2</i> mice (<b><i>H</i></b>) was confirmed by Nissl staining (P29, 2 animals per genotype) (<b><i>G</i></b>). NLL, nuclei of the lateral lemniscus. Dorsal is up, lateral is to the right.</p

Presence of the olivocochlear neurons in <i>Dicer1^Egr2</i> mice.

<p><b><i>A,B</i></b> ChAT immunolabeling was detected throughout the LSO of wild type mice (<b><i>A</i></b>), whereas in <i>Dicer1^Egr2</i> mice, ChAT positive cells were restricted to a densely packed ventrolateral cell group (<b><i>B</i></b>). (<b><i>C–F</i></b>) The same SOC area was labeled by Vglut1, as revealed in the overlay (<b><i>E–F</i></b>). Two animals aged P15–P20 were analyzed per genotype. LSO, lateral superior olive; OCN, olivocochlear neurons. Dorsal is up, lateral is to the right.</p

Quantitative RT-PCR analysis of miR-96 in mouse brainstem.

<p>qRT-PCR analysis reveals that expression of miR-96 increases in the mouse brainstem with age, when comparing E18, P0 and P25. At least 3 biological repeats were done in triplicates (<i>t</i>-test,* P<0.05, ** P<0.01).</p

Smaller size of <i>Dicer1^Egr2</i> mice. Compared to wild type littermates, <i>Dicer1^Egr2</i> mice (black arrows) were smaller and the difference in size increased with age.

<p>P, postnatal day.</p