Subtle Deregulation of the Wnt-Signaling Pathway Through Loss of Apc2 Reduces the Fitness of Intestinal Stem Cells.

The importance of the Wnt-signaling pathway on the regulation and maintenance of the intestinal stem cell (ISC) population is well recognized. However, our current knowledge base is founded on models using systems of gross deregulation of the Wnt-signaling pathway. Given the importance of this signaling pathway on intestinal homeostasis, there is a need to explore the role of more subtle alterations in Wnt-signaling levels within this tissue. Herein, we have used a model of Apc2 loss to meet this aim. Apc2 is a homolog of Apc which can also form a destruction complex capable of binding ß-catenin, albeit less efficiently than Apc. We show that systemic loss of Apc2 results in an increase in the number of cells displaying nuclear ß-catenin at the base of the intestinal crypt. This subsequently impacts the expression levels of several ISC markers and the fitness of ISCs as assessed by organoid formation efficiency. This work provides the first evidence that the function and fitness of ISCs can be altered by even minor misregulation of the Wnt-signaling pathway. Our data highlights the importance of correct maintenance of this crucial signaling pathway in the maintenance and function of the ISC population. Stem Cells 2018;36:114-122.

Brg1 loss attenuates aberrant wnt-signalling and prevents wnt-dependent tumourigenesis in the murine small intestine.

Tumourigenesis within the intestine is potently driven by deregulation of the Wnt pathway, a process epigenetically regulated by the chromatin remodelling factor Brg1. We aimed to investigate this interdependency in an in vivo setting and assess the viability of Brg1 as a potential therapeutic target. Using a range of transgenic approaches, we deleted Brg1 in the context of Wnt-activated murine small intestinal epithelium. Pan-epithelial loss of Brg1 using VillinCreERT2 and AhCreERT transgenes attenuated expression of Wnt target genes, including a subset of stem cell-specific genes and suppressed Wnt-driven tumourigenesis improving animal survival. A similar increase in survival was observed when Wnt activation and Brg1 loss were restricted to the Lgr5 expressing intestinal stem cell population. We propose a mechanism whereby Brg1 function is required for aberrant Wnt signalling and ultimately for the maintenance of the tumour initiating cell compartment, such that loss of Brg1 in an Apc-deficient context suppresses adenoma formation. Our results highlight potential therapeutic value of targeting Brg1 and serve as a proof of concept that targeting the cells of origin of cancer may be of therapeutic relevance.

Cited1 deficiency suppresses intestinal tumorigenesis.

Conditional deletion of Apc in the murine intestine alters crypt-villus architecture and function. This process is accompanied by multiple changes in gene expression, including upregulation of Cited1, whose role in colorectal carcinogenesis is unknown. Here we explore the relevance of Cited1 to intestinal tumorigenesis. We crossed Cited1 null mice with Apc(Min/+) and AhCre(+)Apc(fl/fl) mice and determined the impact of Cited1 deficiency on tumour growth/initiation including tumour multiplicity, cell proliferation, apoptosis and the transcriptome. We show that Cited1 is up-regulated in both human and murine tumours, and that constitutive deficiency of Cited1 increases survival in Apc(Min/+) mice from 230.5 to 515 days. However, paradoxically, Cited1 deficiency accentuated nearly all aspects of the immediate phenotype 4 days after conditional deletion of Apc, including an increase in cell death and enhanced perturbation of differentiation, including of the stem cell compartment. Transcriptome analysis revealed multiple pathway changes, including p53, PI3K and Wnt. The activation of Wnt through Cited1 deficiency correlated with increased transcription of ß-catenin and increased levels of dephosphorylated ß-catenin. Hence, immediately following deletion of Apc, Cited1 normally restrains the Wnt pathway at the level of ß-catenin. Thus deficiency of Cited1 leads to hyper-activation of Wnt signaling and an exaggerated Wnt phenotype including elevated cell death. Cited1 deficiency decreases intestinal tumourigenesis in Apc(Min/+) mice and impacts upon a number of oncogenic signaling pathways, including Wnt. This restraint imposed by Cited1 is consistent with a requirement for Cited1 to constrain Wnt activity to a level commensurate with optimal adenoma formation and maintenance, and provides one mechanism for tumour repression in the absence of Cited1.

Evidence for a crucial role of paneth cells in mediating the intestinal response to injury.

The identification of the intestinal stem cell (ISC) markers Lgr5 and Bmi-1 has furthered our understanding of how they accomplish homeostasis in this rapidly self-renewing tissue. Recent work indicates that these markers identify a cycling Lgr5(+) ISC which can be replaced by a quiescent Bmi-1(+) ISC. Currently, there is little data on how these cells interact to control intestinal crypt homeostasis and regeneration. This interaction likely involves other differentiated cells within the niche as it has previously been demonstrated that the "stemness" of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. To investigate this, we used two conditional mouse models to delete the transcription factor ß-catenin within the intestinal crypt. Critically these differ in their ability to drive recombination within Paneth cells and therefore allow us to compare the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. After gene deletion, the intestines in the model in which Paneth cells were retained showed a rapid recovery and repopulation of the crypt-villus axis presumably from either a spared ISC or the hypothetical quiescent ISCs. However, in the absence of Paneth cells the recovery ability was compromised resulting in complete loss of intestinal epithelial integrity. This data indicates that the Paneth cells play a crucial role within the in vivo ISC niche in aiding recovery following substantial insult.

Brg1 is required for stem cell maintenance in the murine intestinal epithelium in a tissue-specific manner.

Brg1 is a chromatin remodeling factor involved in mediation of a plethora of signaling pathways leading to its participation in various physiological processes both during development and in adult tissues. Among other signaling pathways, the Wnt pathway has been proposed to require Brg1 for transactivation of its target genes. Given the pivotal role of the Wnt pathway in the maintenance of normal intestinal homeostasis, we aimed to investigate the effects of Brg1 loss on the intestinal physiology. To this end, we deleted Brg1 in the murine small and large intestinal epithelia using a range of transgenic approaches. Pan-epithelial loss of Brg1 in the small intestine resulted in crypt ablation, while partial Brg1 deficiency led to gradual repopulation of the intestinal mucosa with wild-type cells. In contrast, Brg1 loss in the large intestinal epithelium was compensated by upregulation of Brm. We propose that while Brg1 is dispensable for the survival and function of the progenitor and differentiated cells in the murine intestinal epithelium, it is essential for the maintenance of the stem cell population in a tissue-specific manner.

Entopic overexpression of Ascl2 does not accelerate tumourigenesis in ApcMin mice.

OBJECTIVE: Expression of the Wnt target gene ASCL2 is elevated in 78% of intestinal neoplasia datasets (Oncomine), suggesting a role for deregulated ASCL2 in the aetiology of intestinal tumourigenesis. Furthermore, ectopic expression of Ascl2 has previously been shown to lead to hyperplasia in the mouse. However, elevated levels of ASCL2 does not have an impact on the overall survival or recurrence-free survival rates in colorectal cancer patients. Here the authors use a novel mouse model to analyse the role of Ascl2 in intestinal tumourigenesis and address the controversy surrounding the relevance of this gene to the aetiology of colorectal cancer. DESIGN: The authors have generated a mouse possessing a transgene carrying the Ascl2 gene together with its endogenous promoter and regulatory regions, thereby elevating Ascl2 expression in an authentic manner. The authors have further intercrossed these Ascl2 overexpressers to the classic Apc(Min) model, to study the consequence of elevated Ascl2 expression in intestinal tumourigenesis. RESULTS: Here the authors genetically demonstrate that elevated expression of Ascl2 in a Wnt signalling dependent manner specifically in the stem cell compartment of the intestine neither increases tumour formation nor diminishes survival in a well established intestinal tumour model, the Apc(min) mouse. CONCLUSION: The authors conclude that ectopic expression of Ascl2 is more important in the aetiology of neoplasia than overexpression of Ascl2.

Epigenetic Regulation of Dlg1, via Kaiso, Alters Mitotic Spindle Polarity and Promotes Intestinal Tumorigenesis.

Both alterations to the epigenome and loss of polarity have been linked to cancer initiation, progression, and metastasis. It has previously been demonstrated that loss of the epigenetic reader protein Kaiso suppresses intestinal tumorigenesis in the Apc+/min mouse model, in which altered polarity plays a key role. Thus, we investigated the link between Kaiso deficiency, polarity, and suppression of intestinal tumorigenesis. We used Kaiso-deficient mice to conditionally delete Apc within the intestinal epithelia and demonstrated upregulation of the spindle polarity genes Dlg1 and Dlgap1. To understand the role of Dlg1, we generated Villin-creApc+/minDlg1flx/flx Kaiso-/y mice to analyze gene expression, survival, tumor burden, and spindle orientation. In vivo analysis of the Dlg1-deficient intestine revealed improper orientation of mitotic spindles and a decreased rate of cellular migration. Loss of Dlg1 decreased survival in Apc+/min mice, validating its role as a tumor suppressor in the intestine. Significantly, the increased survival of Apc+/minKaisoy/- mice was shown to be dependent on Dlg1 expression. Taken together, these data indicate that maintenance of spindle polarity in the intestinal crypt requires appropriate regulation of Dlg1 expression. As Dlg1 loss leads to incorrect spindle orientation and a delay in cells transiting the intestinal crypt. We propose that the delayed exit from the crypt increase the window in which spontaneous mutations can become fixed, producing a "tumor-permissive" environment, without an increase in mutation rate. IMPLICATIONS: Loss of mitotic spindle polarity delays the exit of cells from the intestinal crypt and promotes a tumorigenic environment.

Organoids as a Model for Colorectal Cancer.

Modelling human diseases in in vitro systems is undisputedly an invaluable research tool, yet there are many limitations. Some of those limitations have been overcome through the introduction of organoid culture systems, which have revolutionised colorectal cancer research and enabled an array of new experimental techniques. This 3D system models the physiology, shape, dynamics and cell make-up of the intestinal epithelium producing a relevant and highly adaptable model system. The increased functional relevance of this model compared to the use of 2D cancer cell lines makes it an invaluable tool for both basic and translational research. As the limitations of this system are being overcome to make high-throughput assays possible, it is clear that organoids are becoming a mainstay of colorectal cancer research. This review aims to explore the advantages and limitations of this system and discusses the future directions enabled by this model.

Bootstrap Signal-to-Noise Confidence Intervals: An Objective Method for Subject Exclusion and Quality Control in ERP Studies.

Analysis of event-related potential (ERP) data includes several steps to ensure that ERPs meet an appropriate level of signal quality. One such step, subject exclusion, rejects subject data if ERP waveforms fail to meet an appropriate level of signal quality. Subject exclusion is an important quality control step in the ERP analysis pipeline as it ensures that statistical inference is based only upon those subjects exhibiting clear evoked brain responses. This critical quality control step is most often performed simply through visual inspection of subject-level ERPs by investigators. Such an approach is qualitative, subjective, and susceptible to investigator bias, as there are no standards as to what constitutes an ERP of sufficient signal quality. Here, we describe a standardized and objective method for quantifying waveform quality in individual subjects and establishing criteria for subject exclusion. The approach uses bootstrap resampling of ERP waveforms (from a pool of all available trials) to compute a signal-to-noise ratio confidence interval (SNR-CI) for individual subject waveforms. The lower bound of this SNR-CI (SNRLB ) yields an effective and objective measure of signal quality as it ensures that ERP waveforms statistically exceed a desired signal-to-noise criterion. SNRLB provides a quantifiable metric of individual subject ERP quality and eliminates the need for subjective evaluation of waveform quality by the investigator. We detail the SNR-CI methodology, establish the efficacy of employing this approach with Monte Carlo simulations, and demonstrate its utility in practice when applied to ERP datasets.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models.

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the ''stemness'' of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

What are the best routes to effectively model human colorectal cancer?

Colorectal cancer (CRC) is the third most common cancer in the UK, with over 37,500 people being diagnosed every year. Survival rates for CRC have doubled in the last 30 years and it is now curable if diagnosed early, but still over half of all sufferers do not survive for longer than 5 years after diagnosis. The major complication to treating this disease is that of metastasis, specifically to the liver, which is associated with a 5 year survival of less than 5%. These statistics highlight the importance of the development of earlier detection techniques and more targeted therapeutics. The future of treating this disease therefore lies in increasing understanding of the mutations which cause tumourigenesis, and insight into the development and progression of this complex disease. This can only be achieved through the use of functional models which recapitulate all aspects of the human disease. There is a wide range of models of CRC available to researchers, but all have their own strengths and weaknesses. Here we review how CRC can be modelled and discuss the future of modelling this complex disease, with a particular focus on how genetically engineered mouse models have revolutionised this area of research.