Spatiotemporal reprogramming of differentiated cells underlies regeneration and neoplasia in the intestinal epithelium.

Although the mammalian intestinal epithelium manifests robust regenerative capacity after various cytotoxic injuries, the underlying mechanism has remained unclear. Here we identify the cyclin-dependent kinase inhibitor p57 as a specific marker for a quiescent cell population located around the +4 position of intestinal crypts. Lineage tracing reveals that the p57+ cells serve as enteroendocrine/tuft cell precursors under normal conditions but dedifferentiate and act as facultative stem cells to support regeneration after injury. Single-cell transcriptomics analysis shows that the p57+ cells undergo a dynamic reprogramming process after injury that is characterized by fetal-like conversion and metaplasia-like transformation. Population-level analysis also detects such spatiotemporal reprogramming widely in other differentiated cell types. In intestinal adenoma, p57+ cells manifest homeostatic stem cell activity, in the context of constitutively activated spatiotemporal reprogramming. Our results highlight a pronounced plasticity of the intestinal epithelium that supports maintenance of tissue integrity in normal and neoplastic contexts.

High Yap and Mll1 promote a persistent regenerative cell state induced by Notch signaling and loss of p53.

Specified intestinal epithelial cells reprogram and contribute to the regeneration and renewal of the epithelium upon injury. Mutations that deregulate such renewal processes may contribute to tumorigenesis. Using intestinal organoids, we show that concomitant activation of Notch signaling and ablation of p53 induce a highly proliferative and regenerative cell state, which is associated with increased levels of Yap and the histone methyltransferase Mll1. The induced signaling system orchestrates high proliferation, self-renewal, and niche-factor-independent growth, and elevates the trimethylation of histone 3 at lysine 4 (H3K4me3). We demonstrate that Yap and Mll1 are also elevated in patient-derived colorectal cancer (CRC) organoids and control growth and viability. Our data suggest that Notch activation and p53 ablation induce a signaling circuitry involving Yap and the epigenetic regulator Mll1, which locks cells in a proliferative and regenerative state that renders them susceptible for tumorigenesis.

Intestinal epithelial N-acylphosphatidylethanolamine phospholipase D links dietary fat to metabolic adaptations in obesity and steatosis.

Variations in N-acylethanolamines (NAE) levels are associated with obesity and metabolic comorbidities. Their role in the gut remains unclear. Therefore, we generated a mouse model of inducible intestinal epithelial cell (IEC)-specific deletion of N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD), a key enzyme involved in NAE biosynthesis (Napepld∆IEC). We discovered that Napepld∆IEC mice are hyperphagic upon first high-fat diet (HFD) exposure, and develop exacerbated obesity and steatosis. These mice display hypothalamic Pomc neurons dysfunctions and alterations in intestinal and plasma NAE and 2-acylglycerols. After long-term HFD, Napepld∆IEC mice present reduced energy expenditure. The increased steatosis is associated with higher gut and liver lipid absorption. Napepld∆IEC mice display altered gut microbiota. Akkermansia muciniphila administration partly counteracts the IEC NAPE-PLD deletion effects. In conclusion, intestinal NAPE-PLD is a key sensor in nutritional adaptation to fat intake, gut-to-brain axis and energy homeostasis and thereby constitutes a novel target to tackle obesity and related disorders.

Laminin α1 orchestrates VEGFA functions in the ecosystem of colorectal carcinoma.

BACKGROUND INFORMATION: Tumor stroma remodeling is a key feature of malignant tumors and can promote cancer progression. Laminins are major constituents of basement membranes that physically separate the epithelium from the underlying stroma. RESULTS: By employing mouse models expressing high and low levels of the laminin α1 chain (LMα1), we highlighted its implication in a tumor-stroma crosstalk, thus leading to increased colon tumor incidence, angiogenesis and tumor growth. The underlying mechanism involves attraction of carcinoma-associated fibroblasts by LMα1, VEGFA expression triggered by the complex integrin α2ß1-CXCR4 and binding of VEGFA to LM-111, which in turn promotes angiogenesis, tumor cell survival and proliferation. A gene signature comprising LAMA1, ITGB1, ITGA2, CXCR4 and VEGFA has negative predictive value in colon cancer. CONCLUSIONS: Together, we have identified VEGFA, CXCR4 and α2ß1 integrin downstream of LMα1 in colon cancer as of bad prognostic value for patient survival. SIGNIFICANCE: This information opens novel opportunities for diagnosis and treatment of colon cancer.

HIF-1α Deletion in the Endothelium, but Not in the Epithelium, Protects From Radiation-Induced Enteritis.

BACKGROUND & AIMS: Radiation therapy in the pelvic area is associated with side effects that impact the quality of life of cancer survivors. Interestingly, the gastrointestinal tract is able to adapt to significant changes in oxygen availability, suggesting that mechanisms related to hypoxia sensing help preserve tissue integrity in this organ. However, hypoxia-inducible factor (HIF)-dependent responses to radiation-induced gut toxicity are unknown. Radiation-induced intestinal toxicity is a complex process involving multiple cellular compartments. Here, we investigated whether epithelial or endothelial tissue-specific HIF-1α deletion could affect acute intestinal response to radiation. METHODS: Using constitutive and inducible epithelial or endothelial tissue-specific HIF-1α deletion, we evaluated the consequences of epithelial or endothelial HIF-1α deletion on radiation-induced enteritis after localized irradiation. Survival, radiation-induced tissue injury, molecular inflammatory profile, tissue hypoxia, and vascular injury were monitored. RESULTS: Surprisingly, epithelium-specific HIF-1α deletion does not alter radiation-induced intestinal injury. However, irradiated VECad-Cre+/-HIF-1αFL/FL mice present with lower radiation-induced damage, showed a preserved vasculature, reduced hypoxia, and reduced proinflammatory response compared with irradiated HIF-1αFL/FL mice. CONCLUSIONS: We demonstrate in vivo that HIF-1α impacts radiation-induced enteritis and that this role differs according to the targeted cell type. Our work provides a new role for HIF-1α and endothelium-dependent mechanisms driving inflammatory processes in gut mucosae. Results presented show that effects on normal tissues have to be taken into account in approaches aiming to modulate hypoxia or hypoxia-related molecular mechanisms.

Colorectal cancer specific conditions promote Streptococcus gallolyticus gut colonization.

Colonization by Streptococcus gallolyticus subsp. gallolyticus (SGG) is strongly associated with the occurrence of colorectal cancer (CRC). However, the factors leading to its successful colonization are unknown, and whether SGG influences the oncogenic process or benefits from the tumor-prone environment to prevail remains an open question. Here, we elucidate crucial steps that explain how CRC favors SGG colonization. By using mice genetically prone to CRC, we show that SGG colonization is 1,000-fold higher in tumor-bearing mice than in normal mice. This selective advantage occurs at the expense of resident intestinal enterococci. An SGG-specific locus encoding a bacteriocin ("gallocin") is shown to kill enterococci in vitro. Importantly, bile acids strongly enhance this bacteriocin activity in vivo, leading to greater SGG colonization. Constitutive activation of the Wnt pathway, one of the earliest signaling alterations in CRC, and the decreased expression of the bile acid apical transporter gene Slc10A2, as an effect of the Apc founding mutation, may thereby sustain intestinal colonization by SGG. We conclude that CRC-specific conditions promote SGG colonization of the gut by replacing commensal enterococci in their niche.

Hemidesmosome integrity protects the colon against colitis and colorectal cancer.

OBJECTIVE: Epidemiological and clinical data indicate that patients suffering from IBD with long-standing colitis display a higher risk to develop colorectal high-grade dysplasia. Whereas carcinoma invasion and metastasis rely on basement membrane (BM) disruption, experimental evidence is lacking regarding the potential contribution of epithelial cell/BM anchorage on inflammation onset and subsequent neoplastic transformation of inflammatory lesions. Herein, we analyse the role of the α6ß4 integrin receptor found in hemidesmosomes that attach intestinal epithelial cells (IECs) to the laminin-containing BM. DESIGN: We developed new mouse models inducing IEC-specific ablation of α6 integrin either during development (α6ΔIEC) or in adults (α6ΔIEC-TAM). RESULTS: Strikingly, all α6ΔIEC mutant mice spontaneously developed long-standing colitis, which degenerated overtime into infiltrating adenocarcinoma. The sequence of events leading to disease onset entails hemidesmosome disruption, BM detachment, IL-18 overproduction by IECs, hyperplasia and enhanced intestinal permeability. Likewise, IEC-specific ablation of α6 integrin induced in adult mice (α6ΔIEC-TAM) resulted in fully penetrant colitis and tumour progression. Whereas broad-spectrum antibiotic treatment lowered tissue pathology and IL-1ß secretion from infiltrating myeloid cells, it failed to reduce Th1 and Th17 response. Interestingly, while the initial intestinal inflammation occurred independently of the adaptive immune system, tumourigenesis required B and T lymphocyte activation. CONCLUSIONS: We provide for the first time evidence that loss of IECs/BM interactions triggered by hemidesmosome disruption initiates the development of inflammatory lesions that progress into high-grade dysplasia and carcinoma. Colorectal neoplasia in our mouse models resemble that seen in patients with IBD, making them highly attractive for discovering more efficient therapies.

Zinc Transporter SLC39A7/ZIP7 Promotes Intestinal Epithelial Self-Renewal by Resolving ER Stress.

Zinc transporters play a critical role in spatiotemporal regulation of zinc homeostasis. Although disruption of zinc homeostasis has been implicated in disorders such as intestinal inflammation and aberrant epithelial morphology, it is largely unknown which zinc transporters are responsible for the intestinal epithelial homeostasis. Here, we show that Zrt-Irt-like protein (ZIP) transporter ZIP7, which is highly expressed in the intestinal crypt, is essential for intestinal epithelial proliferation. Mice lacking Zip7 in intestinal epithelium triggered endoplasmic reticulum (ER) stress in proliferative progenitor cells, leading to significant cell death of progenitor cells. Zip7 deficiency led to the loss of Olfm4+ intestinal stem cells and the degeneration of post-mitotic Paneth cells, indicating a fundamental requirement for Zip7 in homeostatic intestinal regeneration. Taken together, these findings provide evidence for the importance of ZIP7 in maintenance of intestinal epithelial homeostasis through the regulation of ER function in proliferative progenitor cells and maintenance of intestinal stem cells. Therapeutic targeting of ZIP7 could lead to effective treatment of gastrointestinal disorders.

Plasticity of the brush border - the yin and yang of intestinal homeostasis.

The brush border on the apical surface of enterocytes is a highly specialized structure well-adapted for efficient digestion and nutrient transport, whilst at the same time providing a protective barrier for the intestinal mucosa. The brush border is constituted of a densely ordered array of microvilli, protrusions of the plasma membrane, which are supported by actin-based microfilaments and interacting proteins and anchored in an apical network of actomyosin and intermediate filaments, the so-called terminal web. The highly dynamic, specialized apical domain is both an essential partner for the gut microbiota and an efficient signalling platform that enables adaptation to physiological stimuli from the external and internal milieu. Nevertheless, genetic alterations or various pathological stresses, such as infection, inflammation, and mechanical or nutritional alterations, can jeopardize this equilibrium and compromise intestinal functions. Long-time neglected, the intestinal brush-border shall be enlightening again as the central actor of the complex but essential intestinal homeostasis. Here, we review the processes and components involved in brush border organization and discuss pathological mechanisms that can induce brush border defects and their physiological consequences.

Mathematical Modelling of Molecular Pathways Enabling Tumour Cell Invasion and Migration.

Understanding the etiology of metastasis is very important in clinical perspective, since it is estimated that metastasis accounts for 90% of cancer patient mortality. Metastasis results from a sequence of multiple steps including invasion and migration. The early stages of metastasis are tightly controlled in normal cells and can be drastically affected by malignant mutations; therefore, they might constitute the principal determinants of the overall metastatic rate even if the later stages take long to occur. To elucidate the role of individual mutations or their combinations affecting the metastatic development, a logical model has been constructed that recapitulates published experimental results of known gene perturbations on local invasion and migration processes, and predict the effect of not yet experimentally assessed mutations. The model has been validated using experimental data on transcriptome dynamics following TGF-ß-dependent induction of Epithelial to Mesenchymal Transition in lung cancer cell lines. A method to associate gene expression profiles with different stable state solutions of the logical model has been developed for that purpose. In addition, we have systematically predicted alleviating (masking) and synergistic pairwise genetic interactions between the genes composing the model with respect to the probability of acquiring the metastatic phenotype. We focused on several unexpected synergistic genetic interactions leading to theoretically very high metastasis probability. Among them, the synergistic combination of Notch overexpression and p53 deletion shows one of the strongest effects, which is in agreement with a recent published experiment in a mouse model of gut cancer. The mathematical model can recapitulate experimental mutations in both cell line and mouse models. Furthermore, the model predicts new gene perturbations that affect the early steps of metastasis underlying potential intervention points for innovative therapeutic strategies in oncology.

Insertion of a Stent in Obstructive Colon Cancer Can Induce a Metastatic Process in an Experimental Murine Model.

BACKGROUND: Colonic self-expanding metallic stents (SEMS) are used in obstructive colorectal cancer patients as a bridge to surgery. However, its oncologic safety remains uncertain. Therefore, we attempted to clarify this further with an experimental study and constructed a mouse model of colonic cancer. METHODS: CT26 cells were injected in the rectal wall, and to mimic SEMS, a cardiac stent was inserted under endoscopy in occlusive (75 % lumen occlusion) tumors. We set up a control group (n = 22) and a stent group (n = 16), and the findings were compared. We focused on serum lactate dehydrogenase (LDH) concentrations, circulating tumor cells, survival time, peritoneal carcinomatosis, liver metastases, and bioluminescence. RESULTS: One week after stent insertion, the serum LDH concentrations were significantly higher in the stent group (506 ± 203 IU/L) compared to the controls (229 ± 52 IU/L) (P = 0.005). The average survival time before sacrifice was significantly lower in the stent group (15.2 ± 1 days) compared to the controls (20 ± 5 days) (P = 0.005). The presence of a peritoneal carcinomatosis was more frequently observed in the stent group (75 %) than in the controls (50 %). Liver metastases were observed in 19 % of the stent group compared to the controls (4.5 %) (P = 0.29). After multivariate analysis, the stent group was still found to be associated with significantly lower survival time (P = 0.002). CONCLUSIONS: These observations led us to conclude that in our mouse model, SEMS resulted in an increased metastatic process and a shorter survival time. We suggest, therefore, that the utmost caution be exercised when opting for a stent as a bridge to surgery.

Mechanical induction of the tumorigenic ß-catenin pathway by tumour growth pressure.

The tumour microenvironment may contribute to tumorigenesis owing to mechanical forces such as fibrotic stiffness or mechanical pressure caused by the expansion of hyper-proliferative cells. Here we explore the contribution of the mechanical pressure exerted by tumour growth onto non-tumorous adjacent epithelium. In the early stage of mouse colon tumour development in the Notch(+)Apc(+/1638N) mouse model, we observed mechanistic pressure stress in the non-tumorous epithelial cells caused by hyper-proliferative adjacent crypts overexpressing active Notch, which is associated with increased Ret and ß-catenin signalling. We thus developed a method that allows the delivery of a defined mechanical pressure in vivo, by subcutaneously inserting a magnet close to the mouse colon. The implanted magnet generated a magnetic force on ultra-magnetic liposomes, stabilized in the mesenchymal cells of the connective tissue surrounding colonic crypts after intravenous injection. The magnetically induced pressure quantitatively mimicked the endogenous early tumour growth stress in the order of 1,200 Pa, without affecting tissue stiffness, as monitored by ultrasound strain imaging and shear wave elastography. The exertion of pressure mimicking that of tumour growth led to rapid Ret activation and downstream phosphorylation of ß-catenin on Tyr654, imparing its interaction with the E-cadherin in adherens junctions, and which was followed by ß-catenin nuclear translocation after 15 days. As a consequence, increased expression of ß-catenin-target genes was observed at 1 month, together with crypt enlargement accompanying the formation of early tumorous aberrant crypt foci. Mechanical activation of the tumorigenic ß-catenin pathway suggests unexplored modes of tumour propagation based on mechanical signalling pathways in healthy epithelial cells surrounding the tumour, which may contribute to tumour heterogeneity.

Suppressing TGFß signaling in regenerating epithelia in an inflammatory microenvironment is sufficient to cause invasive intestinal cancer.

Genetic alterations in the TGFß signaling pathway in combination with oncogenic alterations lead to cancer development in the intestines. However, the mechanisms of TGFß signaling suppression in malignant progression of intestinal tumors have not yet been fully understood. We have examined Apc(Δ716) Tgfbr2(ΔIEC) compound mutant mice that carry mutations in Apc and Tgfbr2 genes in the intestinal epithelial cells. We found inflammatory microenvironment only in the invasive intestinal adenocarcinomas but not in noninvasive benign polyps of the same mice. We thus treated simple Tgfbr2(ΔIEC) mice with dextran sodium sulfate (DSS) that causes ulcerative colitis. Importantly, these Tgfbr2(ΔIEC) mice developed invasive colon cancer associated with chronic inflammation. We also found that TGFß signaling is suppressed in human colitis-associated colon cancer cells. In the mouse invasive tumors, macrophages infiltrated and expressed MT1-MMP, causing MMP2 activation. These results suggest that inflammatory microenvironment contributes to submucosal invasion of TGFß signaling-repressed epithelial cells through activation of MMP2. We further found that regeneration was impaired in Tgfbr2(ΔIEC) mice for intestinal mucosa damaged by DSS treatment or X-ray irradiation, resulting in the expansion of undifferentiated epithelial cell population. Moreover, organoids of intestinal epithelial cells cultured from irradiated Tgfbr2(ΔIEC) mice formed "long crypts" in Matrigel, suggesting acquisition of an invasive phenotype into the extracellular matrix. These results, taken together, indicate that a simple genetic alteration in the TGFß signaling pathway in the inflamed and regenerating intestinal mucosa can cause invasive intestinal tumors. Such a mechanism may play a role in the colon carcinogenesis associated with inflammatory bowel disease in humans.

Apical invasion of intestinal epithelial cells by Salmonella typhimurium requires villin to remodel the brush border actin cytoskeleton.

Salmonella invasion of intestinal epithelial cells requires extensive, though transient, actin modifications at the site of bacterial entry. The actin-modifying protein villin is present in the brush border where it participates in the constitution of microvilli and in epithelial restitution after damage through its actin-severing activity. We investigated a possible role for villin in Salmonella invasion. The absence of villin, which is normally located at the bacterial entry site, leads to a decrease in Salmonella invasion. Villin is necessary for early membrane-associated processes and for optimal ruffle assembly by balancing the steady-state level of actin. The severing activity of villin is important for Salmonella invasion in vivo. The bacterial phosphatase SptP tightly regulates villin phosphorylation, while the actin-binding effector SipA protects F-actin and counterbalances villin-severing activity. Thus, villin plays an important role in establishing the balance between actin polymerization and actin severing to facilitate the initial steps of Salmonella entry.

Intestinal epithelial MyD88 is a sensor switching host metabolism towards obesity according to nutritional status.

Obesity is associated with a cluster of metabolic disorders, low-grade inflammation and altered gut microbiota. Whether host metabolism is controlled by intestinal innate immune system and the gut microbiota is unknown. Here we report that inducible intestinal epithelial cell-specific deletion of MyD88 partially protects against diet-induced obesity, diabetes and inflammation. This is associated with increased energy expenditure, an improved glucose homeostasis, reduced hepatic steatosis, fat mass and inflammation. Protection is transferred following gut microbiota transplantation to germ-free recipients. We also demonstrate that intestinal epithelial MyD88 deletion increases anti-inflammatory endocannabinoids, restores antimicrobial peptides production and increases intestinal regulatory T cells during diet-induced obesity. Targeting MyD88 after the onset of obesity reduces fat mass and inflammation. Our work thus identifies intestinal epithelial MyD88 as a sensor changing host metabolism according to the nutritional status and we show that targeting intestinal epithelial MyD88 constitutes a putative therapeutic target for obesity and related disorders.

Concomitant Notch activation and p53 deletion trigger epithelial-to-mesenchymal transition and metastasis in mouse gut.

Epithelial-to-mesenchymal transition-like (EMT-like) is a critical process allowing initiation of metastases during tumour progression. Here, to investigate its role in intestinal cancer, we combine computational network-based and experimental approaches to create a mouse model with high metastatic potential. Construction and analysis of this network map depicting molecular mechanisms of EMT regulation based on the literature suggests that Notch activation and p53 deletion have a synergistic effect in activating EMT-like processes. To confirm this prediction, we generate transgenic mice by conditionally activating the Notch1 receptor and deleting p53 in the digestive epithelium (NICD/p53(-/-)). These mice develop metastatic tumours with high penetrance. Using GFP lineage tracing, we identify single malignant cells with mesenchymal features in primary and metastatic tumours in vivo. The development of such a model that recapitulates the cellular features observed in invasive human colorectal tumours is appealing for innovative drug discovery.

Conditional expression of fascin increases tumor progression in a mouse model of intestinal cancer.

While absent from normal epithelia, an actin bundling protein, fascin, becomes expressed in invasive carcinoma of different origins. It is highly enriched at the tumors' invasive front suggesting that it could play a role in cancer invasion. Multiple studies have shown that fascin, through its role in formation of cellular protrusions such as filopodia and invadopodia, enhances cancer cell migration and invasion in vitro. However, the role of fascin in vivo remains unknown. We have generated a compound transgenic mouse model that allows expression of fascin in the intestinal epithelium in the Apc-mutated background. Conditional expression of fascin led to decrease in mice survival and increase in tumor burden compared to control animals. Induction of fascin expression in adult tumor-bearing animals accelerated tumor progression and led to formation of invasive adenocarcinoma. Altogether, our study shows that fascin can promote tumor progression in vivo, but also unravels an unexpected role of fascin in tumor initiation.

Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture.

The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here we used a single air-liquid interface culture method without modification to engineer oncogenic mutations into primary epithelial and mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia as a result of expression of Kras carrying the G12D mutation (Kras(G12D)), p53 loss or both and readily generated adenocarcinoma after in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, Kras(G12D) and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), as compared to the more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the IGF2 (insulin-like growth factor-2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.

Oncogenic mutations in intestinal adenomas regulate Bim-mediated apoptosis induced by TGF-ß.

In the majority of microsatellite-stable colorectal cancers (CRCs), an initiating mutation occurs in the adenomatous polyposis coli (APC) or ß-catenin gene, activating the ß-catenin/TCF pathway. The progression of resulting adenomas is associated with oncogenic activation of KRas and inactivation of the p53 and TGF-ß/Smad functions. Most established CRC cell lines contain mutations in the TGF-ß/Smad pathway, but little is known about the function of TGF-ß in the early phases of intestinal tumorigenesis. We used mouse and human ex vivo 3D intestinal organoid cultures and in vivo mouse models to study the effect of TGF-ß on the Lgr5(+) intestinal stem cells and their progeny in intestinal adenomas. We found that the TGF-ß-induced apoptosis in Apc-mutant organoids, including the Lgr5(+) stem cells, was mediated by up-regulation of the BH3-only proapoptotic protein Bcl-2-like protein 11 (Bim). BH3-mimetic compounds recapitulated the effect of Bim not only in the adenomas but also in human CRC organoids that had lost responsiveness to TGF-ß-induced apoptosis. However, wild-type intestinal crypts were markedly less sensitive to TGF-ß than Apc-mutant adenomas, whereas the KRas oncogene increased resistance to TGF-ß via the activation of the Erk1/2 kinase pathway, leading to Bim down-regulation. Our studies identify Bim as a critical mediator of TGF-ß-induced apoptosis in intestinal adenomas and show that the common progression mutations modify Bim levels and sensitivity to TGF-ß during intestinal adenoma development.