Probes for INS

Recently three different cyclin-dependent kinase 4 and 6 (CDK4/6) dual inhibitors were approved for the treatment of breast cancer (palbociclib, ribociclib and abemaciclib), all of which offer comparable therapeutic benefits. Their safety profiles however are different. For example, neutropenia is observed at varying incidences in patients treated with these drugs; however it is the most common adverse event for palbociclib and ribociclib, whereas diarrhea is the most common adverse event observed in patients treated with abemaciclib. In order to understand the mechanism of diarrhea observed with these drugs and in an effort to guide the development of safer drugs, we compared the effects of oral administration of palbociclib, ribociclib and abemaciclib on the gastrointestinal tract of rats using doses intended to produce comparable CDK4/6 inhibition. Rats administered abemaciclib, but not palbociclib or ribociclib, had fecal alterations, unique histopathological findings and distinctive changes in intestinal gene expression. Morphologic changes in the intestine were characterized by proliferation of crypt cells, loss of goblet cells, poorly differentiated and degenerating enterocytes with loss of microvilli and mucosal inflammation. In the jejunum of abemaciclib-treated rats, down-regulation of enterocyte membrane transporters and up-regulation of genes associated with cell proliferation were observed, consistent with activation of the Wnt pathway and downstream transcriptional regulation. Among these CDK4/6 inhibitors, intestinal toxicity was unique to rats treated with abemaciclib, suggesting a mechanism of toxicity not due to primary pharmacology (CDK4/6 inhibition), but to activity at secondary pharmacological targets.

In the adult mouse spinal cord, the ependymal cell population that surrounds the central canal is thought to be a promising source of quiescent stem cells to treat spinal cord injury. Relatively little is known about the cellular origin of ependymal cells during spinal cord development, or the molecular mechanisms that regulate ependymal cells during adult homeostasis. Using genetic lineage tracing based on the Wnt target gene Axin2, we have characterized Wnt-responsive cells during spinal cord development. Our results revealed that Wnt-responsive progenitor cells are restricted to the dorsal midline throughout spinal cord development, which gives rise to dorsal ependymal cells in a spatially restricted pattern. This is contrary to previous reports that suggested an exclusively ventral origin of ependymal cells, suggesting that ependymal cells may retain positional identities in relation to their neural progenitors. Our results further demonstrated that in the postnatal and adult spinal cord, all ependymal cells express the Wnt/β-catenin signaling target gene Axin2, as well as Wnt ligands. Genetic elimination of β-catenin or inhibition of Wnt secretion in Axin2-expressing ependymal cells in vivo both resulted in impaired proliferation, indicating that Wnt/β-catenin signaling promotes ependymal cell proliferation. These results demonstrate the continued importance of Wnt/β-catenin signaling for both ependymal cell formation and regulation. By uncovering the molecular signals underlying the formation and regulation of spinal cord ependymal cells, our findings thus enable further targeting and manipulation of this promising source of quiescent stem cells for therapeutic interventions.

Abstract

Wnt signaling plays an important role in uterine organogenesis and oncogenesis. Our mRNA expression data documents the expression of various Wnt pathway members during the key stages of uterine epithelial gland development. Our data illustrates the expression of Wnt signaling inhibitors (Axin2, Sfrp2, Sfrp4, Dkk1 and Dkk3) in mice uteri at postnatal day 6 (PND 6) and day 15 (PND 15). They also describe the expression pattern of the Wnt ligands (Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt5b, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a and Wnt10b) in mice uteri with or without progesterone treatment. Detailed interpretation and discussion of these data is presented in the research article entitled “Differential Wnt signaling activity limits epithelial gland development to the anti-mesometrial side of the mouse uterus” [1].

Reactive oxygen species (ROS) participate in numerous cell responses, including proliferation, DNA damage, and cell death. Based on these disparate activities, both promotion and inhibition of ROS have been proposed for cancer therapy. However, how the ROS response is determined is not clear. We examined the activities of ROS in a model of Apc deletion, where loss of the Wnt target gene Myc both rescues APC loss and prevents ROS accumulation. Following APC loss, Myc has been shown to up-regulate RAC1 to promote proliferative ROS through NADPH oxidase (NOX). However, APC loss also increased the expression of TIGAR, which functions to limit ROS. To explore this paradox, we used three-dimensional (3D) cultures and in vivo models to show that deletion of TIGAR increased ROS damage and inhibited proliferation. These responses were suppressed by limiting damaging ROS but enhanced by lowering proproliferative NOX-derived ROS. Despite having opposing effects on ROS levels, loss of TIGAR and RAC1 cooperated to suppress intestinal proliferation following APC loss. Our results indicate that the pro- and anti-proliferative effects of ROS can be independently modulated in the same cell, with two key targets in the Wnt pathway functioning to integrate the different ROS signals for optimal cell proliferation.

The epithelial lining of the Fallopian tube is vital for fertility, providing nutrition to gametes, and facilitating their transport. It is composed of two major cell types: secretory cells and ciliated cells. Interestingly, human ovarian cancer precursor lesions are primarily consisting of secretory cells. It is unclear why secretory cells are the dominant cell type in these lesions. Additionally, the underlying mechanisms governing Fallopian tube epithelial homoeostasis are currently unknown. In the present study, we showed that across the different developmental stages of mouse oviduct, secretory cells are the most frequently dividing cells of the oviductal epithelium. In vivo genetic cell lineage tracing showed that secretory cells not only self-renew, but also give rise to ciliated cells. Analysis of a Wnt reporter mouse model and different Wnt target genes showed that the Wnt signaling pathway is involved in oviductal epithelial homoeostasis. By developing two triple transgenic mouse models, we showed that Wnt/β-catenin signaling is essential for self-renewal as well as differentiation of secretory cells. In summary, our results provide mechanistic insight into oviductal epithelial homoeostasis.

Different thresholds of Wnt signalling are thought to drive stem cell maintenance, regeneration, differentiation and cancer. However, the principle that oncogenic Wnt signalling could be specifically targeted remains controversial. Here we examine the requirement of BCL9/9l, constituents of the Wnt-enhanceosome, for intestinal transformation following loss of the tumour suppressor APC. Although required for Lgr5+ intestinal stem cells and regeneration, Bcl9/9l deletion has no impact upon normal intestinal homeostasis. Loss of BCL9/9l suppressed many features of acute APC loss and subsequent Wnt pathway deregulation in vivo. This resulted in a level of Wnt pathway activation that favoured tumour initiation in the proximal small intestine (SI) and blocked tumour growth in the colon. Furthermore, Bcl9/9l deletion completely abrogated β-catenin driven intestinal and hepatocellular transformation. We speculate these results support the just-right hypothesis of Wnt-driven tumour formation. Importantly, loss of BCL9/9l is particularly effective at blocking colonic tumourigenesis and mutations that most resemble those that occur in human cancer.

Dravet syndrome (DS) is a form of epilepsy with a high incidence of sudden unexpected death in epilepsy (SUDEP). Respiratory failure is a leading cause of SUDEP, and DS patients' frequently exhibit disordered breathing. Despite this, mechanisms underlying respiratory dysfunction in DS are unknown. We found that mice expressing a DS-associated Scn1a missense mutation (A1783V) conditionally in inhibitory neurons (Slc32a1cre/+::Scn1aA1783V fl/+; defined as Scn1aΔE26) exhibit spontaneous seizures, die prematurely and present a respiratory phenotype including hypoventilation, apnea, and a diminished ventilatory response to CO2. At the cellular level in the retrotrapezoid nucleus (RTN), we found inhibitory neurons expressing the Scn1a A1783V variant are less excitable, whereas glutamatergic chemosensitive RTN neurons, which are a key source of the CO2/H+-dependent drive to breathe, are hyper-excitable in slices from Scn1aΔE26 mice. These results show loss of Scn1a function can disrupt respiratory control at the cellular and whole animal levels.