Colorectal Cancer Through Simulation and Experiment

Colorectal cancer has continued to generate a huge amount of research interest over several decades, forming a canonical example of tumourigenesis since its use in Fearon and Vogelstein’s linear model of genetic mutation. Over time, the field has witnessed a transition from solely experimental work to the inclusion of mathematical biology and computer-based modelling. The fusion of these disciplines has the potential to provide valuable insights into oncologic processes, but also presents the challenge of uniting many diverse perspectives. Furthermore, the cancer cell phenotype defined by the ‘Hallmarks of Cancer’ has been extended in recent times and provides an excellent basis for future research. We present a timely summary of the literature relating to colorectal cancer, addressing the traditional experimental findings, summarising the key mathematical and computational approaches, and emphasising the role of the Hallmarks in current and future developments. We conclude with a discussion of interdisciplinary work, outlining areas of experimental interest which would benefit from the insight that mathematical and computational modelling can provide

Hippo pathway effector YAP1 is a regulator of intestinal epithelial cell differentiation

Les cellules souches primordiales, qui sont situées à la base de la crypte dans l'intestin des mammifères, fournissent toutes les cellules épithéliales situées dans les cryptes et les villosités, comme les cellules absorbantes, les cellules caliciformes, les cellules de Paneth et les cellules entéroendocrines. La survie des cellules souches est associée à la niche qui est composée de divers signaux et facteurs provenant des cellules épithéliales et du mésenchyme environnant. Un signal important dont le rôle dans le maintien et la prolifération des cellules souches a été démontré est la voie de signalisation Hippo. Les signaux en amont, la cascade kinases et les gènes cibles en aval composent les trois parties de la voie de signalisation d'Hippo. Cette voie limite la croissance des cellules et la taille des organes par la régulation de YAP1 et TAZ qui sont les composants du coeur de la kinase. La voie active d'Hippo contribue à la phosphorylation et à l'inactivation des YAP1/TAZ, ce qui conduit à leur dégradation ou leur séquestration cytoplasmique. En l'absence de la voie Hippo, YAP1/TAZ actifs passent dans le noyau et induisent la transcription des gènes impliqués dans la croissance cellulaire. La présente étude vise à évaluer l'effet de YAP1 sur la différenciation des cellules épithéliales intestinales. Ainsi, l'expression de YAP1 a été neutralisée dans la cellule HT29 à l'aide de l'ARN interférant. Ensuite, l'immunoblot, l'immunofluorescence indirecte et l'analyse RT-qPCR ont été utilisées pour caractériser les cellules absorbantes et sécrétoires. Les résultats ont montré que l'expression des marqueurs de cellules absorbantes SI et DPPIV et des marqueurs de cellules caliciformes MUC2 et TFF3 a été augmentée de manière significative au niveau transcriptionnel et protéique par l'ablation de YAP1. La formation des cellules de type absorbante et de type caliciforme a été confirmée par immunofluorescence indirecte et microscopie électronique à transmission. De plus, l'expression de deux marqueurs de cellules souches, LGR5 et PROM1, a été réduite de manière significative dans la cellule HT29 déplétée en YAP1 par rapport au contrôle. Ensuite, le mécanisme par lequel YAP1 contrôle la différenciation de la cellule épithéliale intestinale a été étudié. Dans ce contexte, l'expression des facteurs de transcription CDX2, ATOH1, HES1, KLF4 et HNF1α a été mesurée à l'aide du qPCR. Les résultats ont montré une augmentation de CDX2 et ATOH1 ainsi qu'une légère augmentation du facteur de transcription HES1 dans la cellule HT29 déplétées en YAP1. L'augmentation de l'expression de la protéine CDX2 a été confirmée par immunobuvardage, tandis qu'ATOH1 n'était pas détectable. L'effet de CDX2 sur la différenciation des cellules absorbantes et caliciformes a été confirmé par l'ablation de CDX2 dans les cellules déficientes en YAP1. L'ablation de CDX2 dans ces cellules s'est accompagnée du retour à des niveaux de base d'expression de SI, MUC2 et TFF3 de même que de LGR5 et PROM1 comparables aux cellules contrôles. Cependant, l'expression de DPPIV est demeurée élevée suggérant l'implication d'autres facteurs. L'expression de MUC2 est également contrôlée par le facteur de transcription ATOH1. L'induction de l'expression d'ATOH1 dans la cellule HT29 a conduit à une augmentation de l'expression de MUC2. De plus, nos études d’inhibition des membres de la famille de kinase Src montrent une réduction de l’expression de YAP1 qui résulte à une augmentation de l’expression des marqueurs des cellules absorbante et à mucus et de CDX2. Nous pouvons donc conclure que YAP1 régule négativement la différenciation des cellules absorbantes et des cellules caliciformes par l'inhibition du facteur de transcription CDX2.Abstract : LGR5+ crypt base columnar (CBC) stem cells that are located at the base of the crypt in the mammalian intestine, provide all the epithelial cells located in the crypts and villi including absorptive, goblet, Paneth and enteroendocrine cells. The maintenance of LGR5+ stem cells is associated with the niche that is composed of various signals and factors originating from the epithelial cells and surrounding mesenchyme. One important signal which has been reported to have a role in stem cell maintenance and proliferation is the Hippo signaling pathway. Upstream signals, a kinase core and downstream target genes compose the three parts of the Hippo pathway. This pathway restricts cell growth and organ size through regulation of YAP1 and TAZ, which are kinase core components. An active Hippo pathway contributes to the phosphorylation and inactivation of the YAP1/TAZ which leads to its degradation or cytoplasmic sequestration. In the absence of the Hippo pathway, active YAP1/TAZ passes into the nucleus and induces the transcription of genes involved in cell growth. The present study is aimed at the investigation of the effect of YAP1 on intestinal epithelial cell differentiation. Thus, YAP1 expression was knocked down in HT29 cells using shRNA. Then Western blot, immunofluorescence and RT-qPCR analysis were used for the characterization of absorptive and secretory cells. The results showed that the expression of the absorptive cell markers SI and DPPIV, and goblet cell markers MUC2 and TFF3 was increased significantly at both transcriptional and protein levels by YAP1 ablation. The formation of absorptive-like and goblet-like cells was confirmed using indirect immunofluorescence and transmission electron microscopy. Furthermore, the expression of the two stem cell markers LGR5 and PROM1 was decreased significantly in YAP1 knockdown HT29 cells compared with the control. Then the mechanism by which YAP1 controls the differentiation of the intestinal epithelial cell was studied. In this context, the expression of the CDX2, ATOH1, HES1, KLF4 and HNF1α transcription factors was measured using qPCR. The results showed an augmentation of CDX2 and ATOH1, and also a slight increase of the HES1 transcription factor in YAP1 knockdown HT29 cells. An increased expression of CDX2 protein was confirmed by Western blot analysis while ATOH1 expression was not at a detectable level. The effect of CDX2 on absorptive and goblet cell differentiation was confirmed by CDX2 ablation in shYAP1-expressing HT29 cells. CDX2 ablation in shYAP1-expressing HT29 cells was accompanied by a return of SI, MUC2 and TFF3 as well as LGR5 and PROM1 to the expression levels of control HT29 cells. However, DPPIV expression remained high suggesting the involvement of additional transcription factors. The expression of MUC2 may also be controlled by the ATOH1 transcription factor. Induction of ATOH1 expression in HT29 cells resulted in increased MUC2 expression. Furthermore, inhibition of Src family kinases led to a decrease in YAP1 expression as well as an increase in absorptive and goblet cells markers and CDX2 expression. Altogether, this investigation showed that YAP1 negatively regulates the differentiation of both absorptive and goblet cells through the inhibition of the main transcription factor, CDX2

Cross-talk between Hippo and Wnt signalling pathways in intestinal crypts : insights from an agent-based model

Intestinal crypts are responsible for the total cell renewal of the lining of the intestines; this turnover is governed by the interplay between signalling pathways and the cell cycle. The role of Wnt signalling in cell proliferation and differentiation in the intestinal crypt has been extensively studied, with increased signalling found towards the lower regions of the crypt. Recent studies have shown that the Wnt signalling gradient found within the crypt may arise as a result of division-based spreading from a Wnt ‘reservoir’ at the crypt base. The discovery of the Hippo pathway’s involvement in maintaining crypt homeostasis is more recent; a mechanistic understanding of Hippo pathway dynamics, and its possible cross-talk with the Wnt pathway, remains lacking. To explore how the interplay between these pathways may control crypt homeostasis, we extended an ordinary differential equation model of the Wnt signalling pathway to include a phenomenological description of Hippo signalling in single cells, and then coupled it to a cell-based description of cell movement, proliferation and contact inhibition in agent-based simulations. Furthermore, we compared an imposed Wnt gradient with a division-based Wnt gradient model. Our results suggest that Hippo signalling affects the Wnt pathway by reducing the presence of free cytoplasmic β-catenin, causing cell cycle arrest. We also show that a division-based spreading of Wnt can form a Wnt gradient, resulting in proliferative dynamics comparable to imposed-gradient models. Finally, a simulated APC double mutant, with misregulated Wnt and Hippo signalling activity, is predicted to cause monoclonal conversion of the crypt

Stem cell impairment at the host–microbiota interface in colorectal cancer

Colorectal cancer (CRC) initiation is believed to result from the conversion of normal intestinal stem cells (ISCs) into cancer stem cells (CSCs), also known as tumor-initiating cells (TICs). Hence, CRC evolves through the multiple acquisition of well-established genetic and epigenetic alterations with an adenoma–carcinoma sequence progression. Unlike other stem cells elsewhere in the body, ISCs cohabit with the intestinal microbiota, which consists of a diverse community of microorganisms, including bacteria, fungi, and viruses. The gut microbiota communicates closely with ISCs and mounting evidence suggests that there is significant crosstalk between host and microbiota at the ISC niche level. Metagenomic analyses have demonstrated that the host– microbiota mutually beneficial symbiosis existing under physiologic conditions is lost during a state of pathological microbial imbalance due to the alteration of microbiota composition (dysbiosis) and/or the genetic susceptibility of the host. The complex interaction between CRC and microbiota is at the forefront of the current CRC research, and there is growing attention on a possible role of the gut microbiome in the pathogenesis of CRC through ISC niche impairment. Here we primarily review the most recent findings on the molecular mechanism underlying the complex interplay between gut microbiota and ISCs, revealing a possible key role of microbiota in the aberrant reprogramming of CSCs in the initiation of CRC. We also discuss recent advances in OMICS approaches and single-cell analyses to explore the relationship between gut microbiota and ISC/CSC niche biology leading to a desirable implementation of the current precision medicine approaches

SIGNALLING TISSUE RENEWAL AND CRYPT SURVIVAL IN THE HUMAN COLONIC EPITHELIUM AND BARRETT’S OESOPHAGUS

Abstract Stem cell driven tissue renewal in the intestinal epithelium is a tightly regulated and controlled process. The colonic epithelium is organised into millions of invaginations called crypts, each of which represents the self-renewing unit of the tissue. In the mouse, renewal of the intestinal epithelium is regulated by signalling cross-talk between the Wnt, Notch, EGF and TGFβ/BMP pathways. The molecular mechanisms that regulate the processes of tissue renewal in the human are of great interest because they are disrupted in colorectal cancer and inflammatory diseases. Barrett’s oesophagus is an intestinal metaplasia arising in response to inflammation and ulceration provoked by gastroesophageal reflux. Detailed knowledge of the processes and signalling pathways involved in tissue renewal in Barrett’s oesophagus is still lacking and is required to understand more fully the risk and pathogenesis of this metaplasia and oesophageal adenocarcinoma. Intact human colonic crypts were isolated and placed into 3D tissue culture conditions optimised for steady-state tissue renewal. The role of Wnt and TGFβ/BMP signalling pathways in tissue renewal was investigated. Native human colonic crypts exhibited distinct activation profiles for canonical Wnt, TGFβ and BMP pathways. A population of intestinal Lgr5/OLFM4+ stem cells were found to be interspersed between goblet cells at the base of the crypt. Exogenous Wnt signals maintained Lgr5/OLFM4+ stem cells, whilst BMP and TGFβ inhibited and caused complete loss of stem cells. Wnt signals also rescued the inhibitory effects of Dkk1, IWP2 and dnTCF4 on Wnt target gene expression, cell proliferation and crypt length. BMP and TGFβ inhibited Wnt target gene expression, cell proliferation and crypt length. A near-native human Barrett’s oesophagus ex vivo culture model was developed similar to the colonic model which was amenable to real-time time-lapse microscopy and imaging techniques. The Wnt and NFκB signalling pathways exhibited distinct activation profiles. A population of OLFM4+ stem cells were found to reside in the lower third of the Barrett’s crypt. Steady-state tissue renewal in the human colonic epithelium is dependant on Wnt signals combined with suppressed TGFβ/BMP pathways. The human colonic crypt model and the Barrett’s oesophagus crypt model will permit functional interrogation of the mechanisms underlying tissue renewal and risk of inflammatory diseases and adenocarcinoma. *[N.B.: A DVD was attached to this thesis at the time of its submission. Please refer to the author for further details

Transcriptional regulation of intestinal epithelial homeostasis and regeneration

Intestinal stem cells (ISCs) reside at the bottom of intestinal crypts. They divide and give rise to early progenitor cells (+4/+5 cells) when entering the transitamplifying zone (TA), and eventually differentiate into various mature epithelial cell types. Wnt, Notch and Tgf-β/Bmp signalling pathways form gradient of expression along the crypt-villus axis and play a central role in regulating ISC homeostasis and lineage commitment. Despite the good understanding of the signalling pathways in regulating ISC self-renewal and fate decision, the underlying mechanism of the dynamic lineage selection and plasticity of the +4/+5 early progenitors remains largely unknown.Here, we identify Arid3a as a novel regulator of intestinal epithelial cell differentiation and maturation. Arid3a is expressed at the early progenitors at +4/+5 cell positions and most differentiated cells with an expression gradient that accumulates at the tip of the villus. We show that Wnt signalling has an inhibitory role on the expression of Arid3a, while Tgf-β signalling promotes its expression. Intestinal epithelial-specific deletion of Arid3a leads to decreased numbers of proliferating TA cells at the upper crypt and a reduction of Wnt signalling which is associated with a moderate decrease of Paneth cell numbers. Interestingly, ISC numbers are not affected. Most importantly, loss of Arid3a perturbs the zonation programme of the entire intestinal epithelium. Expression analysis of the Arid3a cKO intestine showed a reduced gene signatures of committed cells at the upper crypt and a strong enrichment of mid-villus to villus-tip gene signatures of enterocytes, goblet, enteroendocrine and tuft cells. Our findings suggest that TA cells enter their differentiated states earlier at the expense of their proliferative capacity. Finally, using an irradiation mouse model, we show that loss of Arid3a impairs the regenerative process by altering the dynamics of proliferation and apoptosis.We conclude that Arid3a drives maintenance of epithelial homeostasis across the crypt-villus axis and supports the regenerative capacity of the intestinal epithelium. Our work provides an important advancement in understanding the regulation of TA cells state and the transdifferentiation process in the intestinal epithelium

Dissecting the effect of EGF starvation on the signaling and transcriptomic landscapes of the mouse intestinal epithelium

Die EGFR-Signalübertragung steuert viele verschiedene zelluläre Prozesse in allen Arten von Epithelzellen, einschließlich des Darmepithels. Diese Prozesse reichen von Proliferation und Wachstum über Differenzierung bis hin zu Autophagie und Apoptose. Die vorliegende Studie zielt darauf ab, die Signalveränderungen zu charakterisieren, die im Darmepithel als Reaktion auf EGF-induzierten Hungerstress stattfinden. Kontraintuitiv führte eine 24-stündige EGF-Starre zu einer deutlichen Phosphorylierung von EGFR, MEK1/2 und ERK1/2, was auf eine Aktivierung dieser Signalachse in Darmzellen hindeutet. Diese Veränderungen waren am signifikantesten in den undifferenzierten CD44-reichen Krypta-Basiszellen. Interessanterweise war die EGF-Starvation-induzierte ERK1/2-Phosphorylierung mit der Hochregulierung einer Untergruppe von ERK-Zielgenen verbunden, bei denen es sich zumeist um primäre Zielgene handelt. Die Überexpression des EGFR-Liganden HBEGF und des FGFR-Liganden FGF1 in ausgehungerten Zellen könnte für die hungerbedingte Zunahme der MAPK-Aktivität verantwortlich sein, obwohl eine erhöhte Sekretion dieser Liganden durch ausgehungerte Organoide nicht bestätigt werden konnte. Dennoch wird die kompensatorische Ligandensekretion durch die Beobachtung gestützt, dass die erneute Zugabe von EGF zu ausgehungerten Organoiden die pERK1/2-Spiegel auf den Ausgangswert zurücksetzt, was bedeutet, dass EGF mit einem anderen von ausgehungerten Zellen sezernierten Liganden um den EGFR konkurriert. Zusätzlich zu HBEGF wurde festgestellt, dass andere Gene, die für den Schutz, das Überleben und die Regeneration des Darmepithels bekannt sind, in ausgehungerten Organoiden überexprimiert werden, wie z. B. Reg3b. Insgesamt können die in dieser Studie berichteten EGF-induzierten Veränderungen der MAPK-Signalübertragung und der globalen Genexpression als ein überlebensförderndes Programm interpretiert werden, das bevorzugt in Darmstammzellen und frühen Vorläuferzellen aktiviert wird.EGFR signaling drives many different cellular processes in all kinds of epithelial cells including the intestinal epithelium. Such processes range from proliferation and growth to differentiation to autophagy and apoptosis. The present study aims to characterize signaling changes that take place in the intestinal epithelium in response to EGF starvation-induced stress using epithelial organoids derived from the mouse duodenum and human colorectal tumor tissue. Counterintuitively, 24 h EGF starvation induced a prominent phosphorylation of EGFR, MEK1/2 and ERK1/2 indicating an activation of this signaling axis in intestinal cells. These changes were most significant in the undifferentiated CD44-high crypt base cells. Interestingly, EGF starvation-induced ERK1/2 phosphorylation was associated with upregulation of a subset of ERK target genes that were mostly primary-response targets. Overexpression of the EGFR ligand HBEGF and the FGFR ligand FGF1 in starved cells may account for starvation-driven increase in MAPK activity, although an increased secretion of these ligands by starved organoids was not confirmed. Nevertheless, compensatory ligand secretion is still supported by the observation that EGF re-addition to starved organoids restores pERK1/2 levels to baseline which implies that EGF competes for EGFR with some other ligand secreted by starved cells. In addition to HBEGF, other genes known to promote protection, survival and regeneration of the intestinal epithelium were found to be overexpressed in starved organoids such as Reg3b. Collectively, EGF starvation-induced changes in MAPK signaling and global gene expression reported in this study can be interpreted as a pro-survival program that gets activated preferentially in intestinal stem cells and early progenitors

Notch Signaling Pathway in Tooth Shape Variations throughout Evolution

Evolutionary changes in vertebrates are linked to genetic alterations that often affect tooth crown shape, which is a criterion of speciation events. The Notch pathway is highly conserved between species and controls morphogenetic processes in most developing organs, including teeth. Epithelial loss of the Notch-ligand Jagged1 in developing mouse molars affects the location, size and interconnections of their cusps that lead to minor tooth crown shape modifications convergent to those observed along Muridae evolution. RNA sequencing analysis revealed that these alterations are due to the modulation of more than 2000 genes and that Notch signaling is a hub for significant morphogenetic networks, such as Wnts and Fibroblast Growth Factors. The modeling of these tooth crown changes in mutant mice, via a three-dimensional metamorphosis approach, allowed prediction of how Jagged1-associated mutations in humans could affect the morphology of their teeth. These results shed new light on Notch/Jagged1-mediated signaling as one of the crucial components for dental variations in evolution