This report develops an analytically validated chromogenic in situ hybridization (CISH) assay using branched DNA signal amplification (RNAscope) for detecting the expression of the 5' external transcribed spacer (ETS) of the 45S ribosomal (r) RNA precursor in formalin fixed and paraffin embedded (FFPE) human tissues. 5'ETS/45S CISH was performed on standard clinical specimens and tissue microarrays (TMAs) from untreated prostate carcinomas, high-grade prostatic intraepithelial neoplasia (PIN) and matched benign prostatic tissues. Signals were quantified using image analysis software. The 5'ETS rRNA signal was restricted to the nucleolus. The signal was markedly attenuated in cell lines and in prostate tissue slices after pharmacological inhibition of RNA polymerase I (Pol I) using BMH-21 or actinomycin D, and by RNAi depletion of Pol I, demonstrating validity as a measure of Pol I activity. Clinical human prostate FFPE tissue sections and TMAs showed a marked increase in the signal in the presumptive precursor lesion (high-grade prostatic intraepithelial neoplasia/PIN) and invasive adenocarcinoma lesions (p=0.0001 and p=0.0001, respectively) compared with non-neoplastic luminal epithelium. The increase in 5'ETS rRNA signal was present throughout all Gleason scores and pathological stages at radical prostatectomy, with no marked difference among these. This precursor rRNA assay has potential utility for detection of increased rRNA production in various tumor types and as a novel companion diagnostic for clinical trials involving Pol I inhibition.

IMPLICATIONS:

Increased rRNA production, a possible therapeutic target for multiple cancers, can be detected with a new, validated assay that also serves as a pharmacodynamic marker for Pol I inhibitors.

Among the 10 Frizzled (FZD) isoforms belonging to the Class F of G protein-coupled receptors (GPCRs), FZD10 remains the most enigmatic. FZD10 shows homology to FZD4 and FZD9 and was previously implicated in both β-catenin-dependent and -independent signalling. In normal tissue, FZD10 levels are generally very low; however, its upregulation in synovial carcinoma has attracted some attention for therapy. Our findings identify FZD10 as a receptor interacting with and signalling through the heterotrimeric G protein Gα13 but not Gα12, Gαi1, GαoA,Gαs, or Gαq. Stimulation with the FZD agonist WNT induced the dissociation of the Gα13 protein from FZD10, and led to global Gα12/13-dependent cell changes assessed by dynamic mass redistribution measurements. Furthermore, we show that FZD10 mediates Gα12/13activation-dependent induction of YAP/TAZ transcriptional activity. In addition, we show a distinct expression of FZD10 in embryonic CNS endothelial cells at E11.5-E14.5. Given the well-known importance of Gα13 signalling for the development of the vascular system, the selective expression of FZD10 in brain vascular endothelial cells points at a potential role of FZD10-Gα13 signalling in CNS angiogenesis.

Bone metastases remain a serious health concern because of limited therapeutic options. Here, we report that crosstalk between ROR1-HER3 and the Hippo-YAP pathway promotes breast cancer bone metastasis in a long noncoding RNA-dependent fashion. Mechanistically, the orphan receptor tyrosine kinase ROR1 phosphorylates HER3 at a previously unidentified site Tyr1307, following neuregulin stimulation, independently of other ErbB family members. p-HER3 Tyr1307 recruits the LLGL2-MAYA-NSUN6 RNA-protein complex to methylate Hippo/MST1 at Lys59. This methylation leads to MST1 inactivation and activation of YAP target genes in tumour cells, which elicits osteoclast differentiation and bone metastasis. Furthermore, increased ROR1, p-HER3 Tyr1307 and MAYA levels correlate with tumour metastasis and unfavourable outcomes. Our data provide insights into the mechanistic regulation and linkage of the ROR1-HER3 and Hippo-YAP pathway in a cancer-specific context, and also imply valuable therapeutic targets for bone metastasis and possible therapy-resistant tumours.

Place cells in the CA1 region of the hippocampus express location-specific firing despite receiving a steady barrage of heterogeneously tuned excitatory inputs that should compromise output dynamic range and timing. We examined the role of synaptic inhibition in countering the deleterious effects of off-target excitation. Intracellular recordings in behaving mice demonstrate that bimodal excitation drives place cells, while unimodal excitation drives weaker or no spatial tuning in interneurons. Optogenetic hyperpolarization of interneurons had spatially uniform effects on place cell membrane potential dynamics, substantially reducing spatial selectivity. These data and a computational model suggest that spatially uniform inhibitory conductance enhances rate coding in place cells by suppressing out-of-field excitation and by limiting dendritic amplification. Similarly, we observed that inhibitory suppression of phasic noise generated by out-of-field excitation enhances temporal coding by expanding the range of theta phase precession. Thus, spatially uniform inhibition allows proficient and flexible coding in hippocampal CA1 by suppressing heterogeneously tuned excitation.

Certain members of the microbiota genus Bifidobacterium are known to positively influence host well-being. Importantly, reduced bifidobacterial levels are associated with inflammatory bowel disease (IBD) patients, who also have impaired epithelial barrier function, including elevated rates of apoptotic extrusion of small intestinal epithelial cells (IECs) from villi-a process termed 'cell shedding'. Using a mouse model of pathological cell shedding, we show that mice receiving Bifidobacterium breve UCC2003 exhibit significantly reduced rates of small IEC shedding. Bifidobacterial-induced protection appears to be mediated by a specific bifidobacterial surface exopolysaccharide and interactions with host MyD88 resulting in downregulation of intrinsic and extrinsic apoptotic responses to protect epithelial cells under highly inflammatory conditions. Our results reveal an important and previously undescribed role for B. breve, in positively modulating epithelial cell shedding outcomes via bacterial- and host-dependent factors, supporting the notion that manipulation of the microbiota affects intestinal disease outcomes.

Inactivating mutations in the insulin receptor results in extreme insulin resistance. The resulting hyperglycemia is very difficult to treat, and patients are at risk for early morbidity and mortality from complications of diabetes. We used the insulin receptor antagonist S961 to induce severe insulin resistance, hyperglycemia, and ketonemia in mice. Using this model, we show that glucagon receptor (GCGR) inhibition with a monoclonal antibody normalized blood glucose and β-hydroxybutyrate levels. Insulin receptor antagonism increased pancreatic β-cell mass threefold. Normalization of blood glucose levels with GCGR-blocking antibody unexpectedly doubled β-cell mass relative to that observed with S961 alone and 5.8-fold over control. GCGR antibody blockage expanded α-cell mass 5.7-fold, and S961 had no additional effects. Collectively, these data show that GCGR antibody inhibition represents a potential therapeutic option for treatment of patients with extreme insulin-resistance syndromes.

Homeostatic control of breathing, heart rate, and body temperature relies on circuits within the brainstem modulated by the neurotransmitter serotonin (5-HT). Mounting evidence points to specialized neuronal subtypes within the 5-HT system, which have borne out in functional studies, including the modulation of distinct facets of homeostatic control. These functional differences, read out at the organismal level, are likely subserved by differences among 5-HT neuron subtypes at the cellular and molecular levels, including differences in the capacity to co-express other neurotransmitters such as glutamate, GABA, thyrotropin releasing hormone, and substance P encoded by the Tachykinin-1 (Tac1) gene. Here we characterize in mice a 5-HT neuron subtype identified by expression of Tac1 and the transcription factor gene Pet1, thus referred to as the Tac1-Pet1 neuron subtype. Transgenic cell labeling showed Tac1-Pet1 soma resident largely in the caudal medulla. Chemogenetic (CNO-hM4Di) perturbation of Tac1-Pet1 neuron activity resulted in blunting of the respiratory CO2 chemoreflex, which normally augments ventilation in response to hypercapnic acidosis to restore normal pH and PCO2 Tac1-Pet1 axonal boutons were found localized to brainstem areas implicated in respiratory modulation, with highest density in motor nuclei. These findings demonstrate that the activity of a Pet1 neuron subtype with potential to release both 5-HT and substance P is necessary for normal respiratory dynamics, likely via motor outputs that maintain airway patency and engage muscles of respiration. These Tac1-Pet1 neurons may complement the activity of Egr2-Pet1 neurons, previously established in respiratory chemoreception, but which do not innervate respiratory motor nuclei.

SIGNIFICANCE STATEMENT:

5-HT neurons modulate outputs as diverse as body temperature, respiration, aggression, and mood. We characterize a 5-HT neuron subtype defined by expression of Tachykinin1 and Pet1 (Tac1-Pet1 neurons) which projects to respiratory motor nuclei, and when silenced, blunts the ventilatory response to inhaled carbon dioxide. We employ genetic tools to access this subset of 5-HT neurons to query function, anatomy, and connectivity. Localization of synaptic boutons from Tac1-Pet1 neurons, primarily within motor regions, contrasts with those from previously described Egr2-Pet1 neurons, which are chemosensitive and reside in the raphe magnus and project primarily to chemosensory integration, but not motor, regions of the brainstem.

Abstract

Specific inbred strains and transgenic inhibin-α Simian Virus 40 T antigen (inhα/Tag) mice are genetically susceptible to gonadectomy-induced adrenocortical neoplasias. We identified altered gene expression in prepubertally gonadectomized (GDX) inhα/Tag and wild-type (WT) mice. Besides earlier reported Gata4 and Lhcgr, we found up-regulated Esr1, Prlr-rs1, and down-regulated Grb10, Mmp24, Sgcd, Rerg, Gnas, Nfatc2, Gnrhr, Igf2 in inhα/Tag adrenal tumors. Sex-steroidogenic enzyme genes expression (Srd5a1, Cyp19a1) was up-regulated in tumors, but adrenal-specific steroidogenic enzyme (Cyp21a1, Cyp11b1, Cyp11b2) down-regulated. We localized novel Lhcgr transcripts in adrenal cortex parenchyma and in non-steroidogenic A cells, in GDX WT and in intact WT mice. We identified up-regulated Esr1 as a potential novel biomarker of gonadectomy-induced adrenocortical tumors in inhα/Tag mice presenting with an inverted adrenal-to-gonadal steroidogenic gene expression profile. A putative normal adrenal remodeling or tumor suppressor role of the down-regulated genes (e.g. Grb10, Rerg, Gnas, and Nfatc2) in the tumors remains to be addressed.

Abstract

Abstract

Abstract