Probes for INS

Successful embryo implantation requires a receptive endometrium. Poor uterine receptivity can account for implantation failure in women who experience recurrent pregnancy loss or multiple rounds of unsuccessful in vitro fertilization cycles. Here, we demonstrate that the transcription factor Forkhead Box O1 (FOXO1) is a critical regulator of endometrial receptivity in vivo. Uterine ablation of Foxo1 using the progesterone receptor Cre (PgrCre) mouse model resulted in infertility due to altered epithelial cell polarity and apoptosis, preventing the embryo from penetrating the luminal epithelium. Analysis of the uterine transcriptome after Foxo1 ablation identified alterations in gene expression for transcripts involved in the activation of cell invasion, molecular transport, apoptosis, β-catenin (CTNNB1) signaling pathway, and an increase in PGR signaling. The increase of PGR signaling was due to PGR expression being retained in the uterine epithelium during the window of receptivity. Constitutive expression of epithelial PGR during this receptive period inhibited expression of FOXO1 in the nucleus of the uterine epithelium. The reciprocal expression of PGR and FOXO1 was conserved in human endometrial samples during the proliferative and secretory phase. This demonstrates that expression of FOXO1 and the loss of PGR during the window of receptivity are interrelated and critical for embryo implantation.

Fibroblast growth factor (FGF) signaling is essential for many developmental processes and plays a pivotal role in skeletal homeostasis, regeneration and wound healing. FGF signals through one of five tyrosine kinase receptors: Fgfr1a, -1b, -2, -3, -4. To characterize the expression of zebrafish fgfr3 from the larval stage to adulthood, we used RNAscope in situ hybridization on paraffin sections of the zebrafish head. Our study revealed spatial and temporal distribution of fgfr3 transcript in chondrocytes of the head cartilages, osteoblasts involved in bone formation, ventricular zone of the brain, undifferentiated mesenchymal cells of the skin, and lens epithelium of the eye. In general, the expression pattern of zebrafish fgfr3 is similar to the expression observed in higher vertebrates.

While all 2-methylene-19-nor analogs of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) tested produce an increase in epidermal thickness in the rhino mouse, only a subset reduce utricle size (comedolysis). All-trans retinoic acid (atRA) also causes epidermal thickening and a reduction in utricle size in the rhino mouse. We now report that 2-methylene-19-nor-(20S)-1α-hydroxybishomopregnacalciferol (2MbisP), a comedolytic analog, increases epidermal thickening more rapidly than does atRA, while both reduce utricle area at an equal rate. Whereas unlike atRA, 2MbisP does not alter the epidermal growth factor receptor ligand, heparin-binding epidermal growth factor-like growth factor, it does increase the expression of both amphiregulin and epigen mRNA, even after a single dose. In situ hybridization reveals an increase in these transcripts throughout the closing utricle as well as in the interfollicular epidermis. The mRNAs for other EGFR ligands including betacellulin and transforming growth factor-α, as well as the epidermal growth factor receptor are largely unaffected by 2MbisP. Another analog, 2-methylene-19-nor-(20S)-26,27-dimethylene-1α,25-dihydroxyvitamin D3 (CAGE-3), produces epidermal thickening but fails to reduce utricle size or increase AREG mRNA levels. CAGE-3 modestly increases epigen mRNA levels, but only after 5 days of dosing. Thus, 2-MbisP produces unique changes in epidermal growth factor receptor ligand mRNAs that may be responsible for both epidermal proliferation and a reduction in utricle size.

Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.

Pan-fibroblast growth-factor receptor (FGFR) inhibitors hold promise in FGFR-altered patients, but such alterations are rare in advanced urothelial carcinoma. In order to assess whether we may increase the number of eligible patients by using different molecular techniques for detecting alterations, we pooled the results of the centralised FGFR mutation/translocation assays that were performed in Clinical Laboratory Improvement Amendments-certified laboratories within multiple phase 2 trials. At our centre, the same tissue blocks were used to analyse FGFR1-3 messenger RNA expression through messenger RNA in situ hybridisation (ISH; RNAscope 2.5 assay). From October 2016 to March 2017, 52 cases were analysed. Seventeen patients (32.7%) had an upper tract primary tumour. Ten patients (19.2%) had FGFR DNA alterations. Twenty-nine (55.8%) had positive ISH analysis: N=17 score 3, N=12 score 4. Of note, concordance between the two tests was obtained in seven out of 10 patients. Sixty percent of mutated patients had an upper tract primary tumour versus 31% of ISH-positive patients.

PATIENT SUMMARY:

We found three-fold higher frequency of fibroblast growth-factor receptor alterations at the RNA versus DNA level in advanced urothelial carcinoma, with a different distribution according to the method used and the site of the primary tumour. The evaluation of the therapeutic response to pan-fibroblast growth-factor receptor inhibitors according to the method of assessment is warranted.

Abstract

OBJECTIVES:

Metastatic colorectal cancer (CRC) remains a leading cause of cancer related deaths. Patients with oligometastatic liver disease represent a clinical subgroup with heterogeneous course. Until now, biomarkers to characterize outcome and therapeutic options have not been fully established.

METHODS:

We investigated the prevalence of FGFR alterations in a total of 140 primary colorectal tumors and 63 liver metastases of 55 oligometastatic CRC patients. FGF receptors (FGFR1-4) and their ligands (FGF3, 4 and 19) were analyzed for gene amplifications and rearrangements as well as for RNA overexpression in situ. Results were correlated with clinico-pathologic data and molecular subtypes.

RESULTS:

Primary tumors showed FGFR1 (6.3%) and FGF3,4,19 (2.2%) amplifications as well as FGFR1 (10.1%), FGFR2 (5.5%) and FGFR3 (16.2%) overexpression. In metastases, we observed FGFR1 amplifications (4.8%) as well as FGFR1 (8.5%) and FGFR3 (14.9%) overexpression. Neither FGFR2-4 amplifications nor gene rearrangements were observed. FGFR3 overexpression was significantly associated with shorter overall survival in metastases (mOS 19.9 vs. 47.4 months, HR=3.14, p=0.0152), but not in primary CRC (HR=1.01, p=0.985). Although rare, also FGFR1 amplification was indicative of worse outcome (mOS 12.6 vs. 47.4 months, HR=8.83, p=0.00111).

CONCLUSIONS:

We provide the so far most comprehensive analysis of FGFR alterations in primary and metastatic CRC. We describe FGFR3 overexpression in 15% of CRC patients with oligometastatic liver disease as a prognosticator for poor outcome. Recently FGFR3 overexpression has been shown to be a potential therapeutic target. Therefore, we suggest focusing on this subgroup in upcoming clinical trials with FGFR-targeted therapies.

Uterine glands are essential for pregnancy establishment. By employing forkhead box A2 (FOXA2)-deficient mouse models coupled with leukemia inhibitory factor (LIF) repletion, we reveal definitive roles of uterine glands in embryo implantation and stromal cell decidualization. Here we report that LIF from the uterine glands initiates embryo-uterine communication, leading to embryo attachment and stromal cell decidualization. Detailed histological and molecular analyses discovered that implantation crypt formation does not involve uterine glands, but removal of the luminal epithelium is delayed and subsequent decidualization fails in LIF-replaced glandless but not gland-containing FOXA2-deficient mice. Adverse ripple effects of those dysregulated events in the glandless uterus result in embryo resorption and pregnancy failure. These studies provide evidence that uterine glands synchronize embryo-endometrial interactions, coordinate on-time embryo implantation, and impact stromal cell decidualization, thereby ensuring embryo viability, placental growth, and pregnancy success.