Intestinal stem cell dynamics: a story of mice and humans

Stem cell dynamics define the probability of accumulating mutations within the intestinal epithelium. In this issue of Cell Stem Cell, Nicholson et al. (2018) report that human intestinal stem cell dynamics differ significantly from those of mice and establish that oncogenic mutations are more likely to expand; therefore, "normal" epithelium may carry multiple mutations

The central role of Wnt signaling and organoid technology in personalizing anticancer therapy

The Wnt pathway is at the heart of organoid technology, which is set to revolutionize the cancer field. We can now predetermine a patient's response to any given anticancer therapy by exposing tumor organoids established from the patient's own tumor. This cutting-edge biomedical platform translates to patients being treated with the correct drug at the correct dose from the outset, a truly personalized and precise medical approach. A high throughput drug screen on organoids also allows drugs to be tested in limitless combinations. More recently, the tumor cells that are resistant to the therapy given to a patient were selected in culture using the patient's organoids. The resistant tumor organoids were then screened empirically to identify drugs that will kill the resistant cells. This information allows diagnosis in real-time to either prevent tumor recurrence or effectively treat the recurring tumor. Furthermore, the ability to culture stem cell-derived epithelium as organoids has enabled us to begin to understand how a stem cell becomes a cancer cell or to pin-point the genetic alteration that underlies a given genetic syndrome. Here we summarize these advances and the central role of Wnt signaling, and identify the next challenges for organoid technology

Frizzled-7 is required for Wnt signaling in gastric tumours with and without Apc mutations

A subset of patients with gastric cancer have mutations in genes that participate in or regulate Wnt signaling at the level of ligand (Wnt) receptor (Fzd) binding. Moreover, increased Fzd expression is associated with poor clinical outcome. Despite these findings, there are no in vivo studies investigating the potential of targeting Wnt receptors for treating gastric cancer, and the specific Wnt receptor transmitting oncogenic Wnt signaling in gastric cancer is unknown. Here, we use inhibitors of Wnt/Fzd (OMP-18R5/vantictumab) and conditional gene deletion to test the therapeutic potential of targeting Wnt signaling in preclinical models of intestinal-type gastric cancer and ex vivo organoid cultures. Pharmacologic targeting of Fzd inhibited the growth of gastric adenomas in vivo. We identified Fzd7 to be the predominant Wnt receptor responsible for transmitting Wnt signaling in human gastric cancer cells and mouse models of gastric cancer, whereby Fzd7-deficient cells were retained in gastric adenomas but were unable to respond to Wnt signals and consequently failed to proliferate. Genetic deletion of Fzd7 or treatment with vantictumab was sufficient to inhibit the growth of gastric adenomas with or without mutations to Apc. Vantictumab is currently in phase Ib clinical trials for advanced pancreatic, lung, and breast cancer. Our data extend the scope of patients that may benefit from this therapeutic approach as we demonstrate that this drug will be effective in treating patients with gastric cancer regardless of APC mutation status

Expression of R-Spondin 1 in Apc(Min/+) Mice Suppresses Growth of Intestinal Adenomas by Altering Wnt and Transforming Growth Factor Beta Signaling

BACKGROUND & AIMS: Mutations in the APC gene and other genes in the Wnt signaling pathway contribute to development of colorectal carcinomas. R-spondins (RSPOs) are secreted proteins that amplify Wnt signaling in intestinal stem cells. Alterations in RSPO genes have been identified in human colorectal tumors. We studied the effects of RSPO1 overexpression in ApcMin/thorn mutant mice. METHODS: An adeno associated viral vector encoding RSPO1-Fc fusion protein, or control vector, was injected into ApcMin/thornmice. Their intestinal crypts were isolated and cultured as organoids. which were incubated with or without RSPO1-Fc and an inhibitor of transforming growth factor beta receptor (TGFBR). Livers were collected from mice and analyzed by immunohistochemistry. Organoids and adenomas were analyzed by quantitative reverse-transcription PCR, single cell RNA sequencing, and immunohistochemistry. RESULTS: Intestines from Apcthorn/thorn mice injected with the vector encoding RSPO1-Fc had significantly deeper crypts, longer villi, with increased EdU labeling, indicating increased proliferation of epithelial cells, in comparison to mice given control vector. AAV-RSPO1-Fctransduced ApcMin/thorn mice also developed fewer and smaller intestinal tumors and had significantly longer survival times. Adenomas of ApcMin/thorn mice injected with the RSPO1-Fc vector showed a rapid increase in apoptosis and in the expression of Wnt target genes, followed by reduced expression of messenger RNAs and proteins regulated by the Wnt pathway, reduced cell proliferation, and less crypt branching than adenomas of mice given the control vector. Addition of RSPO1 reduced the number of adenoma organoids derived from ApcMin/thorn mice and suppressed expression of Wnt target genes but increased phosphorylation of SMAD2 and transcription of genes regulated by SMAD. Inhibition of TGFBR signaling in organoids stimulated with RSPO1-Fc restored organoid formation and expression of genes regulated by Wnt. The TGFBR inhibitor restored apoptosis in adenomas from ApcMin/thorn mice expressing RSPO1Fc back to the same level as in the adenomas from mice given the control vector. CONCLUSIONS: Expression of RSPO1 in ApcMin/thorn mice increases apoptosis and reduces proliferation and Wnt signaling in adenoma cells, resulting in development of fewer and smaller intestinal tumors and longer mouse survival. Addition of RSPO1 to organoids derived from adenomas inhibits their growth and promotes proliferation of intestinal stem cells that retain the APC protein; these effects are reversed by TGFB inhibitor. Strategies to increase the expression of RSPO1 might be developed for the treatment of intestinal adenomas.Peer reviewe

Retrograde movements determine effective stem cell numbers in the intestine

The morphology and functionality of the epithelial lining differ along the intestinal tract, but tissue renewal at all sites is driven by stem cells at the base of crypts(1-3). Whether stem cell numbers and behaviour vary at different sites is unknown. Here we show using intravital microscopy that, despite similarities in the number and distribution of proliferative cells with an Lgr5 signature in mice, small intestinal crypts contain twice as many effective stem cells as large intestinal crypts. We find that, although passively displaced by a conveyor-belt-like upward movement, small intestinal cells positioned away from the crypt base can function as long-term effective stem cells owing to Wnt-dependent retrograde cellular movement. By contrast, the near absence of retrograde movement in the large intestine restricts cell repositioning, leading to a reduction in effective stem cell number. Moreover, after suppression of the retrograde movement in the small intestine, the number of effective stem cells is reduced, and the rate of monoclonal conversion of crypts is accelerated. Together, these results show that the number of effective stem cells is determined by active retrograde movement, revealing a new channel of stem cell regulation that can be experimentally and pharmacologically manipulated.Peer reviewe

BCL-3 loss sensitises colorectal cancer cells to DNA damage by targeting homologous recombination

The proto-oncogene BCL-3 is upregulated in a subset of colorectal cancers (CRC), where it has been shown to enhance tumour cell survival. However, although increased expression correlates with poor patient prognosis, the role of BCL-3 in determining therapeutic response remains largely unknown. In this study, we use combined approaches in multiple cell lines and pre-clinical mouse models to investigate the function of BCL-3 in the DNA damage response. We show that suppression of BCL-3 increases γH2AX foci formation and decreases homologous recombination in CRC cells, resulting in reduced RAD51 foci number and increased sensitivity to PARP inhibition. Importantly, a similar phenotype is seen in Bcl3-/- mice, where Bcl3-/- mouse crypts also exhibit sensitivity to DNA damage with increased γH2AX foci compared to wild type mice. Additionally, Apc.Kras-mutant x Bcl3-/- mice are more sensitive to cisplatin chemotherapy compared to wild type mice. Taken together, our results identify BCL-3 as a regulator of the cellular response to DNA damage and suggests that elevated BCL-3 expression, as observed in CRC, could increase resistance of tumour cells to DNA damaging agents including radiotherapy. These findings offer a rationale for targeting BCL-3 in CRC as an adjunct to conventional therapies and suggest that BCL-3 expression in tumours could be a useful biomarker in stratification of rectal cancer patients for neo-adjuvant chemoradiotherapy

Epithelial TGFβ engages growth-factor signalling to circumvent apoptosis and drive intestinal tumourigenesis with aggressive features

The pro-tumourigenic role of epithelial TGFβ signalling in colorectal cancer (CRC) is controversial. Here, we identify a cohort of born to be bad early-stage (T1) colorectal tumours, with aggressive features and a propensity to disseminate early, that are characterised by high epithelial cell-intrinsic TGFβ signalling. In the presence of concurrent Apc and Kras mutations, activation of epithelial TGFβ signalling rampantly accelerates tumourigenesis and share transcriptional signatures with those of the born to be bad T1 human tumours and predicts recurrence in stage II CRC. Mechanistically, epithelial TGFβ signalling induces a growth-promoting EGFR-signalling module that synergises with mutant APC and KRAS to drive MAPK signalling that re-sensitise tumour cells to MEK and/or EGFR inhibitors. Together, we identify epithelial TGFβ signalling both as a determinant of early dissemination and a potential therapeutic vulnerability of CRC’s with born to be bad traits

Oncogenic BRAF, unrestrained by TGFβ-receptor signalling, drives right-sided colonic tumorigenesis

Right-sided (proximal) colorectal cancer (CRC) has a poor prognosis and a distinct mutational profile, characterized by oncogenic BRAF mutations and aberrations in mismatch repair and TGFβ signalling. Here, we describe a mouse model of right-sided colon cancer driven by oncogenic BRAF and loss of epithelial TGFβ-receptor signalling. The proximal colonic tumours that develop in this model exhibit a foetal-like progenitor phenotype (Ly6a/Sca1+) and, importantly, lack expression of Lgr5 and its associated intestinal stem cell signature. These features are recapitulated in human BRAF-mutant, right-sided CRCs and represent fundamental differences between left- and right-sided disease. Microbial-driven inflammation supports the initiation and progression of these tumours with foetal-like characteristics, consistent with their predilection for the microbe-rich right colon and their antibiotic sensitivity. While MAPK-pathway activating mutations drive this foetal-like signature via ERK-dependent activation of the transcriptional coactivator YAP, the same foetal-like transcriptional programs are also initiated by inflammation in a MAPK-independent manner. Importantly, in both contexts, epithelial TGFβ-receptor signalling is instrumental in suppressing the tumorigenic potential of these foetal-like progenitor cells

Differential cross section measurements for the production of a W boson in association with jets in proton–proton collisions at √s = 7 TeV

Measurements are reported of differential cross sections for the production of a W boson, which decays into a muon and a neutrino, in association with jets, as a function of several variables, including the transverse momenta (pT) and pseudorapidities of the four leading jets, the scalar sum of jet transverse momenta (HT), and the difference in azimuthal angle between the directions of each jet and the muon. The data sample of pp collisions at a centre-of-mass energy of 7 TeV was collected with the CMS detector at the LHC and corresponds to an integrated luminosity of 5.0 fb[superscript −1]. The measured cross sections are compared to predictions from Monte Carlo generators, MadGraph + pythia and sherpa, and to next-to-leading-order calculations from BlackHat + sherpa. The differential cross sections are found to be in agreement with the predictions, apart from the pT distributions of the leading jets at high pT values, the distributions of the HT at high-HT and low jet multiplicity, and the distribution of the difference in azimuthal angle between the leading jet and the muon at low values.United States. Dept. of EnergyNational Science Foundation (U.S.)Alfred P. Sloan Foundatio