Complex interplay between β-catenin signalling and Notch effectors in intestinal tumorigenesis

International audienceAims The activation of β-catenin signalling is a key step in intestinal tumorigenesis. Interplay between the β-catenin and Notch pathways during tumorigenesis has been reported, but the mechanisms involved and the role of Notch remain unclear. Methods Notch status was analysed by studying expression of the Notch effector Hes1 and Notch ligands/receptors in human colorectal cancer (CRC) and mouse models of Apc mutation. A genetic approach was used, deleting the Apc and RBP-J or Atoh1 genes in murine intestine. CRC cell lines were used to analyse the control of Hes1 and Atoh1 by β-catenin signalling. Results Notch signalling was found to be activated downstream from β-catenin. It was rapidly induced and maintained throughout tumorigenesis. Hes1 induction was mediated by β-catenin and resulted from both the induction of the Notch ligand/receptor and Notch-independent control of the Hes1 promoter by β-catenin. Surprisingly, the strong phenotype of unrestricted proliferation and impaired differentiation induced by acute Apc deletion in the intestine was not rescued by conditional Notch inactivation. Hyperactivation of β-catenin signalling overrode the forced differention induced by Notch inhibition, through the downregulation of Atoh1, a key secretory determinant factor downstream of Notch. This process involves glycogen synthase kinase 3 β (GSK3β) and proteasome-mediated degradation. The restoration of Atoh1 expression in CRC cell lines displaying β-catenin activation was sufficient to increase goblet cell differentiation, whereas genetic ablation of Atoh1 greatly increased tumour formation in Apc mutant mice. Conclusion Notch signalling is a downstream target of β-catenin hyperactivation in intestinal tumorigenesis. However, its inhibition had no tumour suppressor effect in the context of acute β-catenin activation probably due to the downregulation of Atoh1. This finding calls into question the use of γ-secretase inhibitors for the treatment of CRC and suggests that the restoration of Atoh1 expression in CRC should be considered as a therapeutic approach

Intestinal crypts reproducibly expand in culture

In vitro growth techniques for intestinal crypts and single intestinal stem cells have been recently described, but several questions of translational importance remain unaddressed. The purpose of this study was to: first, evaluate if intestinal crypts reproducibly expand in vitro; second, determine the impact of age and region of intestine on crypt growth in vitro; and third, determine the effects of cryopreservation on crypt growth in vitro

Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium

Tuft cells represent a fourth type of intestinal secretory cell that constitutes the primary source of endogenous intestinal opioids and are the only epithelial cell that constitutively express cyclooxygenases

Intestinal stem cells remain viable after prolonged tissue storage

Intestinal stem cells (ISCs) are responsible for renewal of the epithelium both during normal homeostasis and following injury. As such they have significant therapeutic potential. However, it is unknown whether ISCs can survive tissue storage. We hypothesized that, although the majority of epithelial cells may die, ISCs would remain viable for at least 24 h at 4°C. To explore this hypothesis, jejuni of C57Bl6/J or Lgr5-LacZ mice were removed and either processed immediately or placed in phosphate buffered saline (PBS) at 4°C. Delayed isolations of epithelia were performed after 24, 30, or 48 h storage. At the light microscope level, despite extensive apoptosis of villus epithelial cells, small intestinal crypts remained morphologically intact through 30 h and ISCs were identifiable via Lgr5-LacZ positivity. Electron microscopy showed that ISCs retain high integrity through 24 h. When assessed by flow cytometry, ISCs were more resistant to degeneration than the rest of the epithelium, including neighboring Paneth cells, with higher viability across all time points. Culture of isolated crypts showed no loss of capacity to form complex enteroids after 24 h tissue storage, with efficiencies after 7 days of culture remaining above 80%. By 30 h storage, efficiencies declined but budding capability was retained. We conclude that, with delay in isolation, ISCs remain viable and retain their proliferative capacity. In contrast, the remainder of the epithelium, including the Paneth cells, exhibits degeneration and programmed cell death. If these findings are recapitulated with human tissue, storage at 4°C may offer a valuable temporal window for harvest of crypts or ISCs for therapeutic application

Atonal homolog 1 Is a Tumor Suppressor Gene

Colon cancer accounts for more than 10% of all cancer deaths annually. Our genetic evidence from Drosophila and previous in vitro studies of mammalian Atonal homolog 1 (Atoh1, also called Math1 or Hath1) suggest an anti-oncogenic function for the Atonal group of proneural basic helix-loop-helix transcription factors. We asked whether mouse Atoh1 and human ATOH1 act as tumor suppressor genes in vivo. Genetic knockouts in mouse and molecular analyses in the mouse and in human cancer cell lines support a tumor suppressor function for ATOH1. ATOH1 antagonizes tumor formation and growth by regulating proliferation and apoptosis, likely via activation of the Jun N-terminal kinase signaling pathway. Furthermore, colorectal cancer and Merkel cell carcinoma patients show genetic and epigenetic ATOH1 loss-of-function mutations. Our data indicate that ATOH1 may be an early target for oncogenic mutations in tissues where it instructs cellular differentiation

Insulin Concentration Modulates Hepatic Lipid Accumulation in Mice in Part via Transcriptional Regulation of Fatty Acid Transport Proteins

Fatty liver disease (FLD) is commonly associated with insulin resistance and obesity, but interestingly it is also observed at low insulin states, such as prolonged fasting. Thus, we asked whether insulin is an independent modulator of hepatic lipid accumulation.In mice we induced, hypo- and hyperinsulinemia associated FLD by diet induced obesity and streptozotocin treatment, respectively. The mechanism of free fatty acid induced steatosis was studied in cell culture with mouse liver cells under different insulin concentrations, pharmacological phosphoinositol-3-kinase (PI3K) inhibition and siRNA targeted gene knock-down. We found with in vivo and in vitro models that lipid storage is increased, as expected, in both hypo- and hyperinsulinemic states, and that it is mediated by signaling through either insulin receptor substrate (IRS) 1 or 2. As previously reported, IRS-1 was up-regulated at high insulin concentrations, while IRS-2 was increased at low levels of insulin concentration. Relative increase in either of these insulin substrates, was associated with an increase in liver-specific fatty acid transport proteins (FATP) 2&5, and increased lipid storage. Furthermore, utilizing pharmacological PI3K inhibition we found that the IRS-PI3K pathway was necessary for lipogenesis, while FATP responses were mediated via IRS signaling. Data from additional siRNA experiments showed that knock-down of IRSs impacted FATP levels.States of perturbed insulin signaling (low-insulin or high-insulin) both lead to increased hepatic lipid storage via FATP and IRS signaling. These novel findings offer a common mechanism of FLD pathogenesis in states of both inadequate (prolonged fasting) and ineffective (obesity) insulin signaling